WO2022145420A1 - 新規な多層ポリマーコーティング架橋アルギン酸ゲルファイバ - Google Patents

新規な多層ポリマーコーティング架橋アルギン酸ゲルファイバ Download PDF

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WO2022145420A1
WO2022145420A1 PCT/JP2021/048567 JP2021048567W WO2022145420A1 WO 2022145420 A1 WO2022145420 A1 WO 2022145420A1 JP 2021048567 W JP2021048567 W JP 2021048567W WO 2022145420 A1 WO2022145420 A1 WO 2022145420A1
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Prior art keywords
cells
alginic acid
formula
producing
polymer
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PCT/JP2021/048567
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English (en)
French (fr)
Japanese (ja)
Inventor
正司 古迫
直人 津田
勉 佐藤
智裕 鳴海
愼吾 佐藤
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Mochida Pharmaceutical Co Ltd
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Mochida Pharmaceutical Co Ltd
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Priority to KR1020237021149A priority Critical patent/KR20230127997A/ko
Priority to AU2021414759A priority patent/AU2021414759A1/en
Priority to CN202180087547.8A priority patent/CN116710110A/zh
Priority to JP2022573085A priority patent/JPWO2022145420A1/ja
Priority to EP21915279.0A priority patent/EP4268857A4/en
Priority to US18/269,705 priority patent/US20240132854A1/en
Priority to CA3203302A priority patent/CA3203302A1/en
Priority to PCT/JP2022/024935 priority patent/WO2022270549A1/ja
Priority to EP22828465.9A priority patent/EP4361249A4/en
Priority to CN202280044010.8A priority patent/CN117529547A/zh
Priority to AU2022298245A priority patent/AU2022298245A1/en
Priority to KR1020237044096A priority patent/KR20240024839A/ko
Priority to CA3223483A priority patent/CA3223483A1/en
Priority to JP2022568561A priority patent/JPWO2022270549A1/ja
Publication of WO2022145420A1 publication Critical patent/WO2022145420A1/ja
Anticipated expiration legal-status Critical
Priority to JP2023194056A priority patent/JP7450793B2/ja
Priority to US18/392,583 priority patent/US12312574B2/en
Priority to JP2024031804A priority patent/JP7545000B2/ja
Ceased legal-status Critical Current

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Definitions

  • the present invention relates to a polymer-coated crosslinked alginate gel fiber for producing an antibody, a physiologically active substance, etc., a method for producing the fiber, and a method for producing an antibody, a physiologically active substance, etc. using the fiber.
  • the present invention also relates to a multilayer polymer-coated crosslinked alginate gel fiber for producing an antibody, a physiologically active substance, etc., a method for producing the fiber, and a method for producing an antibody, a physiologically active substance, etc. using the fiber.
  • antibodies by culture using animal cells.
  • physiologically active substances eg, interferon, erythropoietin, IL-2 (interleukin-2), CSF (colony stimulating factor), TNF (tumor necrosis factor), etc.
  • IL-2 interleukin-2
  • CSF colony stimulating factor
  • TNF tumor necrosis factor
  • CHO cells derived from Chinese hamster ovary
  • Sp2 / 0 cells, NS0 cells and the like are used as host cells, and in particular, CHO cells are cells capable of suspension culture.
  • CHO cells are cells capable of suspension culture.
  • the cell proliferation rate is high and it is easy to mass-produce the target protein by mass-culturing CHO cells, it is frequently used in the production of antibodies.
  • the antibody-producing cell line is expanded and cultured while controlling the culture conditions such as medium composition, temperature, stirring conditions, gas exchange, and pH, and finally. Culturing is carried out in a large-scale production culture tank with a scale of several thousand to 10,000 L.
  • Patent Document 1 International Publication No. 2011/046105
  • Patent Document 2 Special Patent Document 2: Kai 2016-77229 (Ab.).
  • Patent Document 3 International Publication No. 2020/032221 pamphlet.
  • Patent Document 4 International Publication No. 2015/178427 Pamphlet
  • Patent Document 5 Patent No. 6601931
  • Patent Document 6 Japanese Unexamined Patent Publication No. 2014-236698).
  • Patent Document 9 Japanese Patent Application Laid-Open No. 2014-136128.
  • a cell structure eg, sheet-like in which a mixture containing adherent cells (specifically, C2C12 cells), microcarriers, and gel-like polysaccharides (specifically, alginate gel) is coated with polyamino acids.
  • adherent cells specifically, C2C12 cells
  • microcarriers specifically, microcarriers
  • gel-like polysaccharides specifically, alginate gel
  • Non-Patent Document 1 PA. -Lab Chip, 2008, 8, p1255-1257).
  • Patent Document 11 International Publication No. 2019
  • Patent Document 12 International Publication No. 2021/125255 Pamphlet
  • Patent Documents 1 to 13 and Non-Patent Document 1 describe polymer-coated crosslinked alginate gel fibers or multilayer polymer-coated crosslinked alginate gel fibers for the production of the antibodies, physiologically active substances, etc. of the present invention, methods for producing each gel fiber, and each. A method for producing an antibody or the like using a gel fiber is not disclosed or suggested.
  • Alginate gel fiber containing cells capable of producing antibodies, physiologically active substances, etc., in particular, cell culture can be performed for a long period of time (for example, 7 days or more, 14 days or more, 28 days or more) without decomposition of the fiber.
  • a long period of time for example, 7 days or more, 14 days or more, 28 days or more
  • a novel polymer-coated cross-linked alginate gel fiber for the production of antibodies, physiologically active substances, etc. which is formed by coating a cross-linked alginate gel obtained by performing a cross-linking reaction with an alginic acid derivative with a cationic polymer, and a cross-linked alginate gel fiber thereof. I found a manufacturing method. Further, when cells producing antibodies, physiologically active substances, etc.
  • the crosslinked alginate gel obtained by performing a crosslinking reaction using the chemically modified alginic acid derivatives represented by the formulas (I) and the formula (II) described in the later aspect [1B] is coated with a cationic polymer.
  • Polymer coating for the production of antibodies, physiologically active substances, etc. which is formed by coating a crosslinked alginate gel fiber with an anionic polymer, which is a novel multilayer polymer coating for the production of antibodies, physiologically active substances, etc.
  • a crosslinked alginate gel fiber and a method for producing the same have been found. Further, when the antibody-producing cells were cultured using the multi-layer polymer-coated cross-linked alginate gel fiber, it was found that the multi-layer polymer-coated cross-linked alginate gel fiber could continuously produce antibodies and the like for a long period of time without decomposition. ..
  • the present invention provides a novel polymer-coated crosslinked alginate gel fiber and a method for producing an antibody, a physiologically active substance, or the like using the gel fiber.
  • a mixed solution containing a cell capable of producing an antibody, a physiologically active substance, or the like, a chemically modified alginic acid derivative represented by the formulas (I) and the formula (II) described in the following embodiment [1], and the like is prepared.
  • a polymer-coated crosslinked alginate gel fiber capable of continuously producing an antibody, a physiologically active substance, or the like for a long period of time is provided.
  • the present invention also provides a novel multilayer polymer-coated crosslinked alginate gel fiber and a method for producing an antibody, a physiologically active substance, or the like using the gel fiber.
  • a mixed solution containing a cell capable of producing an antibody, a physiologically active substance or the like, a chemically modified alginic acid derivative represented by the formulas (I) and the formula (II) described in the following embodiment [1B], and the like is prepared.
  • Polymer coating obtained by coating the cross-linked alginic acid gel produced using the above with a cationic polymer By coating the cross-linked alginate gel fiber with an anionic polymer, antibodies, physiologically active substances, etc. can be continuously produced for a long period of time.
  • Multilayer polymer coated crosslinked alginate gel fibers are provided.
  • a cross-linked alginate gel (also referred to as a core layer) formed by using a mixed solution containing a chemically modified alginic acid derivative or the like is obtained from poly-L-ornithine, polyallylamine, polyethyleneimine, or polymethylene-CO-guanidine (PMCG).
  • a polymer-coated crosslinked alginate gel fiber By coating with a cationic polymer (also referred to as a cationic polymer layer), a polymer-coated crosslinked alginate gel fiber can be produced, and when cultured using the fiber, a long period of time (up to 47 in the examples described later) is obtained. It was found that antibodies or insulin could be produced continuously (for days).
  • the polymer-coated crosslinked alginate gel fiber of the present invention provides an environment suitable for cells capable of producing antibodies, physiologically active substances, etc., and the antibodies, physiologically active substances, etc. produced in the core layer of the fiber are the core layer. , Has the characteristic of continuously permeating the cationic polymer layer and being released out of the fiber.
  • the outside of the cationic polymer layer of the polymer-coated crosslinked alginic acid gel fiber containing the tosirizumab-producing CHO cells in the core layer is designated as an anionic polymer (also referred to as an anionic polymer layer), alginic acid, formula.
  • an anionic polymer also referred to as an anionic polymer layer
  • alginic acid formula.
  • the multilayer polymer-coated cross-linked alginate gel fiber of the present invention provides an environment suitable for cells capable of producing antibodies, physiologically active substances, etc., like the above-mentioned polymer-coated cross-linked alginate gel fiber, and is used in the core layer of the fiber.
  • the produced antibody, physiologically active substance, or the like has a characteristic that it continuously permeates the core layer, the cationic polymer layer, and the anionic polymer layer and is released to the outside of the fiber.
  • the polymer-coated crosslinked alginate gel fiber and the multilayer polymer-coated crosslinked alginate gel fiber of the present invention provide an environment suitable for the production of antibodies, physiologically active substances and the like. There is little physical stress on the cells producing antibodies, bioactive substances, etc. encapsulated in the core layer, and it is expected that the encapsulated cells will continue to produce antibodies, bioactive substances, etc. for a long period of time. Therefore, a method for producing an antibody, a physiologically active substance, or the like using such a fiber can be expected to dramatically improve the production efficiency of the antibody, the physiologically active substance, or the like.
  • antibody production unlike the suspension culture of an antibody that requires a large-scale culture tank, it is expected that the antibody will be produced in a small-scale production facility. It is also expected to be a continuous production technology for next-generation antibody drugs suitable for the production of small-quantity and various items of antibody drugs.
  • FIG. 1 It is a schematic diagram explaining one aspect of the manufacturing process of a multilayer polymer coated crosslinked alginate gel fiber. It is a cross section of a multilayer polymer coated crosslinked alginate gel fiber. It is a schematic diagram explaining that the metabolites and waste products such as an antibody, a physiologically active substance, etc. produced in a core layer, and a culture solution (nutrient source) and oxygen permeate through a cationic polymer layer and an anionic polymer layer. It is a photograph before culturing of the polymer-coated crosslinked alginate gel fiber (FB9-3-c3) of (Example F9).
  • FB9-3-c3 polymer-coated crosslinked alginate gel fiber
  • the first aspect is as follows.
  • [Chemically modified alginic acid derivative represented by the formula (I)] The following formula (I): [In formula (I), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; Akn - L1- (Akn represents a cyclic alkyne group;- L1- is a divalent linker that binds to a cyclic alkyne group (Akn)).
  • [Chemically modified alginic acid derivative represented by formula (II)] The following formula (II): [In formula (II), (ALG) represents alginic acid; -NHCO- represents an amide bond via any carboxyl group of alginic acid; -L 2- represents the table below: A chemically modified alginic acid derivative represented by [representing a linker selected from the group consisting of the partial structural formulas (in each formula, the outside of the broken line at both ends is not included)].
  • the first aspect is as follows.
  • a polymer-coated crosslinked alginate gel fiber comprising a core layer and a cationic polymer layer arranged outside the core layer, wherein the core layer is a cell capable of producing an antibody, a physiologically active substance, or the like and formula (I).
  • a polymer-coated crosslinked alginic acid gel fiber comprising a crosslinked alginic acid gel in which a crosslink is formed using a chemically modified alginic acid derivative represented by the formula (II), wherein the cationic polymer layer is a cationic polymer.
  • the chemically modified alginic acid derivative represented by the formula (I) and the formula (II) is the same as that defined in the above aspect [1].
  • the chemically modified alginic acid derivative represented by the formula (I), Akn - L1- is preferably described in the following table: It is a group selected from the group consisting of the partial structural formulas described in (the right side of the broken line is not included in each formula).
  • the chemically modified alginic acid derivative represented by the formula (I), Akn - L1- is more preferably described in the following table: It is a group selected from the group consisting of the partial structural formulas described in (the right side of the broken line is not included in each formula).
  • the chemically modified alginic acid derivative Akn—L1 ⁇ represented by the formula ( I ) is more preferably the following partial structural formula (in each formula). , Does not include the right side of the dashed line): It is a group selected from the group consisting of.
  • Akn-L1- of the chemically modified alginic acid derivative represented by the formula (I) is particularly preferably the following partial structural formula (in each formula). , Does not include the right side of the dashed line): It is a group selected from.
  • Akn-L1- of the chemically modified alginic acid derivative represented by the formula (I) is shown in the following table: It is a group selected from the group consisting of the partial structural formulas described in (the right side of the broken line is not included in each formula); Preferably, the table below: It is a group selected from the group consisting of the partial structural formulas described in (the right side of the broken line is not included in each formula); More preferably, the table below: It is a group selected from the group consisting of the partial structural formulas described in (the right side of the broken line is not included in each formula); More preferably, the following partial structural formula (in each formula, the right side of the broken line is not included): It is a group selected from.
  • the chemically modified alginic acid derivative —L2- represented by the formula ( II ) is preferably described in the following table: It is a group selected from the group consisting of the partial structural formulas described in (the outside of the broken line at both ends is not included in each formula).
  • the chemically modified alginic acid derivative —L2- represented by the formula ( II ) is more preferably described in the following table: It is a group selected from the group consisting of the partial structural formulas described in (the outside of the broken line at both ends is not included in each formula).
  • the chemically modified alginic acid derivative —L2- represented by the formula ( II ) is more preferably the following partial structural formula (in each formula, The outside of the broken line at both ends is not included): It is a group selected from the group consisting of.
  • the chemically modified alginic acid derivative —L2- represented by the formula ( II ) is particularly preferably the following partial structural formula (in each formula, The outside of the broken line at both ends is not included): It is a group selected from the group consisting of.
  • the chemically modified alginic acid derivative ⁇ L2- represented by the formula ( II ) is represented by the following table: It is a group selected from the group consisting of the partial structural formulas described in (the outside of the broken line at both ends is not included in each formula); Preferably, the following partial structural formula (in each formula, the right side of the broken line is not included): It is a group selected from the group consisting of the partial structural formulas described in (the outside of the broken line at both ends is not included in each formula); More preferably, the following partial structural formula (in each formula, the right side of the broken line is not included): It is a group selected from the group consisting of the partial structural formulas described in (the outside of the broken line at both ends is not included in each formula); More preferably, the following partial structural formula (in each formula, the outside of the broken line at both ends is not included): It is a group selected from the group consisting of.
  • [1-2A] Represented by the formulas (I) and (II) in which the definitions of Akn, -L 1- , and -L 2- described in the above embodiments [1] to [1-2-5] are appropriately combined.
  • the chemically modified alginic acid derivative By using the chemically modified alginic acid derivative, a preferred embodiment of the crosslinked alginate gel in the core layer of the polymer-coated crosslinked alginate gel fiber of the present invention can be arbitrarily formed.
  • the cells capable of producing an antibody, a physiologically active substance, etc. contained in the core layer of a polymer-coated crosslinked alginate gel fiber are, for example, an antibody (human antibody, humanized). Selected from the group consisting of cells producing various monoclonal antibodies such as antibodies, chimeric antibodies, mouse antibodies, etc., cells producing physiologically active substances, and cells capable of producing various useful substances useful as raw materials for pharmaceuticals, chemical raw materials, food raw materials, etc. It is a cell.
  • the cells capable of producing an antibody (also referred to as antibody-producing cells) contained in the core layer of the polymer-coated crosslinked alginate gel fiber are hybrid dormas or antibody expression vectors.
  • the cultured cells (host cells) used as the host thereof are, for example, CHO cells, CHO cell substrains, COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, PERC6 cells, and the like.
  • the antibody-producing cells contained in the core layer of the polymer-coated crosslinked alginate gel fiber are preferably host cells such as CHO cells and CHO cell substrains. , COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, and PERC6 cells; more preferably from CHO cells, CHO cell substrains, Sp2 / 0 cells, and NS0 cells. Cells selected from the group; more preferably CHO cells or CHO cell substrains.
  • the antibody-producing cell contained in the core layer of the polymer-coated crosslinked alginate gel fiber is, for example, an antibody-producing CHO cell whose host cell is a CHO cell.
  • an antibody-producing CHO cell whose host cell is a CHO cell.
  • muromonab-CD3-producing CHO cells trussumab-producing CHO cells, rituximab-producing CHO cells, paribismab-producing CHO cells, infliximab-producing CHO cells, baciliximab-producing CHO cells, tosirizumab-producing CHO cells, gemtuzumab-producing CHO cells, bebasimab-producing CHO cells.
  • Ibritumomab-producing CHO cells Ibritumomab-producing CHO cells, adalimumab-producing CHO cells, setuximab-producing CHO cells, ranibizmab-producing CHO cells, omalizumab-producing CHO cells, eculizumab-producing CHO cells, panitzummab-producing CHO cells, ustequinumab-producing CHO cells, golimumab-producing CHO cells, kana , Denosumab-producing CHO cells, Mogamurizumab-producing CHO cells, Celtrizumab-producing CHO cells, Ofatumumab-producing CHO cells, Pertuzumab-producing CHO cells, Brentuccimab-producing CHO cells, Natalizumab-producing CHO cells, Nivormab-producing CHO cells, Alemtuzumab-producing CHO cells, Alemtuzumab-producing CHO cells CHO cells, rams
  • CHO cells selected from the group consisting of HO cells; for example, tocilizumab-producing CHO cells or anti-GPVI antibody-producing CHO cells.
  • the cell capable of producing a physiologically active substance contained in the core layer of the polymer-coated crosslinked alginate gel fiber is, for example, Insulin-secreting cells, pancreatic islets, pancreatic islets cells, dopamine-secreting cells, pituitary cells, growth hormone-secreting cells, parathyroid cells, nerve growth factor-secreting cells, blood coagulation factor-secreting cells, hepatocytes, epithelial body cells, erythropoetin-secreting cells , A cell selected from the group consisting of norepinephrine-secreting cells and the like.
  • the physiologically active substance-producing cells contained in the core layer of the polymer-coated crosslinked islet gel fiber are preferably from insulin-secreting cells, pancreatic islets, and pancreatic islet cells.
  • the components that can be additionally contained in the core layer of the polymer-coated crosslinked alginate gel fiber include, for example, an alginic acid solution, an alginate gel, a medium, a culture solution, and collagen. It is a component selected from the group consisting of solutions, methylcellulose, sucrose solutions and the like.
  • the components that can be additionally contained in the core layer of the polymer-coated crosslinked alginic acid gel fiber are preferably an alginic acid solution, an alginic acid gel, a medium, and an alginic acid solution. It is a component selected from the group consisting of culture medium.
  • the introduction rate of the group (Akn-L 1 -NH 2 groups: Akn-L 1 -is the same as the definition in the above embodiment [1]) is, for example, in the range of about 0.1 to about 30 mol%. It is preferably in the range of about 0.3 to about 20 mol%; more preferably in the range of about 0.5 to about 10 mol%.
  • the introduction rate of the groups (N 3 -L 2 -NH 2 groups: -L 2- is the same as the definition in the above embodiment [1]) is, for example, in the range of about 0.1 to about 30 mol%. It is preferably in the range of about 0.3 to about 20 mol%; more preferably in the range of about 0.5 to about 15 mol%.
  • [1-9] In the above embodiment [1] or [1A], it is used for preparing an alginic acid solution that can be additionally contained in the core layer of a polymer-coated crosslinked alginic acid gel fiber, or an alginic acid solution used for forming an alginic acid gel.
  • the weight average molecular weight of alginic acid eg, sodium alginate, etc.
  • GPC method gel filtration chromatography
  • the weight average molecular weight of the alginic acid used is, for example, in the range of about 150,000 Da to about 2.5 million Da; preferably. Range from about 300,000 Da to about 2.5 million Da; more preferably from about 700,000 Da to about 1,400,000 Da, from about 800,000 Da to about 1,500,000 Da, from about 1,400,000 Da. It is a range selected from 2,000,000 Da, or about 1,500,000 to about 2,500,000.
  • the chemically modified alginate derivative represented by the formula (I) used for forming the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber is, for example, in the range of about 0.01 to about 1.5% by weight; preferably in the range of about 0.05 to about 1.0% by weight; more preferably about 0.08. It is in the range of about 0.75% by weight.
  • the concentration of the solution is, for example, in the range of about 0.01 to about 1.5% by weight; preferably in the range of about 0.05 to about 1.0% by weight; more preferably about 0.08. It is in the range of about 0.75% by weight.
  • the chemically modified alginic acid derivative represented by the formula (I) used for forming the crosslinked alginic acid gel contained in the core layer of the polymer-coated crosslinked alginic acid gel fiber is, for example, in the range of about 0.02 to about 2.0% by weight; preferably about 0.1 to about 2.0% by weight. %; More preferably, it is in the range of about 0.15 to about 1.5% by weight.
  • the concentration of the alginic acid solution that can be additionally contained in the core layer of the polymer-coated crosslinked alginic acid gel fiber or the alginic acid solution used for forming the alginic acid gel is for example, it is in the range of 0 to about 1.98% by weight; preferably in the range of 0 to about 1.8% by weight; more preferably in the range of 0 to about 1.7% by weight.
  • the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber includes an alginate solution or an alginate gel formed from the alginic acid solution.
  • the total concentration of the mixed solution containing the chemically modified arginic acid derivatives represented by the formulas (I) and (II) used for forming the core layer and the concentration of the arginic acid solution is preferably about 0.5 to about 2. It is 0.0% by weight; more preferably, it is a concentration selected from about 1.0% by weight, about 1.5% by weight and about 2.0% by weight.
  • the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber includes an alginate solution or an alginate gel formed from the alginic acid solution.
  • concentration of the mixed solution containing the chemically modified arginic acid derivatives represented by the formulas (I) and the formula (II) used for forming the core layer C1 (% by weight)
  • concentration of the arginic acid solution C2 (% by weight)
  • (C1: C2) (about 0.2: about 1.3), (about 0.5: about 1.0), (about 1.0: about 0.5), ( It is a combination selected from the group consisting of (about 1.5: 0), (about 0.66: about 1.34), and (about 0.34: about 0.66).
  • the cross-linked alginic acid gel contained in the core layer of the polymer-coated cross-linked alginic acid gel fiber includes an alginic acid solution or an alginic acid gel formed from the alginic acid solution.
  • concentration of the solution of the chemically modified alginic acid derivative represented by the formula (I) used for forming the core layer C1A (% by weight)
  • concentration of the solution of the chemically modified alginic acid derivative represented by the formula (II) C1N (C1N (%)).
  • the cross-linked alginic acid gel contained in the core layer of the polymer-coated cross-linked alginic acid gel fiber includes an alginic acid solution or an alginic acid gel formed from the alginic acid solution.
  • the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber has the following formula (III-L): [In formula (III-L), -CONH- and -NHCO- at both ends represent an amide bond mediated by any carboxyl group of alginic acid; -X- is the table below: It is a cyclic group selected from the group of partial structural formulas described in (in each formula, the outside of the broken line at both ends is not included); -L 1- is the table below when -X- is (CL-1) or (CL-1-r).
  • -L 1- is the table below when -X- is (CL-2) or (CL-2-r). It is a divalent linker selected from the group of substructural formulas described in (in each formula, the outside of the broken line at both ends is not included); -L 2 --includes a chemical cross-linking via a group represented by [the same as the definition of formula (II) in the above aspect [1]].
  • the preferred combination of ⁇ L2 - X—L1 ⁇ of the group represented by the formula (III-L) is the formula in the following table: As shown by the partial structure selected from the group of (-L 1 -in the table is the same as the definition of preferred -L 1- described in the above embodiment [1-12-1];-L 2 -Is the same as the definition of preferred -L 2- " described in the above aspect [1-2-1]; -X-is as described in the above aspect [1-12]).
  • the combination of -L 2 -X-L 1- is based on the formula in the table below: As shown by the partial structure selected from the group of (-L 1 -in the table is the same as the more preferred definition of -L 1- described in the above aspect [1-12-1]; 2 -is the same as the more preferred definition of -L2- described in the above aspect [1-2-2]; -X-is as described in the above aspect [1-12]);
  • the combination of -L 2 -X-L 1- is based on the formula in the table below: As shown by the partial structure selected from the group of (-L 1 -in the table is the same as the more preferred definition of -L 1- described in the above aspect [1-12-1]; 2 -is the same as the more preferred definition of -L 2 --described in the embodiment [1-2-3]; -X-is as described in the embodiment [1-12]).
  • the combination of -L 2 -X-L 1- is based on the formula in the table below: As shown by the partial structure selected from the group of (-L 1 -in the table is the same as the definition of particularly preferred -L 1- described in the above embodiment [1-12-1]; 2 -is the same as the definition of particularly preferred -L 2- " described in the above aspect [1-2-4]; -X-is as described in the above aspect [1-12]).
  • the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber is the formula (III-L) [1-12] described in the above aspect [1-12].
  • formula (III-L) -CONH- and -NHCO-, -X- at both ends are the same as the definitions in the above embodiment [1-12]; -1) or (CL-1-r) is the same as the group represented by the partial structural formula (LK-1a) according to the above aspect [1-12]; -X- is (CL-2).
  • (CL- 2 -r) is the same as the group represented by the partial structural formula (LK-2-1) described in the above aspect [1-12]; It is the same as the group selected from the partial structural formulas (LN-1), (LN-3) and (LN-5) described in [1]].
  • —X— is (CL-1) or (CL-1-r)
  • preferred, more preferred, and even more preferred —L1 ⁇ are each.
  • preferable, more preferable, and even more preferable ⁇ L 1 ⁇ are the partial structural formulas (LK-2) according to the above aspect [1-12], respectively.
  • -L 2- is preferably the portion according to the above aspect [1-2-1].
  • the crosslinked alginate gel contained in the core layer is contained in the formulas (III-L) [formula (III-L) of the above-mentioned embodiment [1-12], respectively.
  • the definition is the same as the definition of aspect [1-12]] or in the formulas (III-L) [formula (III-L)] of the aspect [1-12A], each definition is the same as the definition of mode [1-12A]. It is the same as the definition of], and includes chemical cross-linking via a group and ionic cross-linking via a divalent metal ion.
  • the divalent metal ion used for forming an ionic bridge in the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber is preferably calcium ion.
  • Magnesium ion, barium ion, strontium ion and zinc ion more preferably calcium ion, barium ion or strontium ion; more preferably calcium ion or barium ion.
  • the aqueous solution of the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber containing the divalent metal ion used for forming an ion bridge is calcium chloride.
  • An aqueous solution containing a divalent metal ion selected from the group consisting of an aqueous solution, an aqueous solution of calcium carbonate, an aqueous solution of calcium gluconate, an aqueous solution of barium chloride, an aqueous solution of strontium chloride and the like can be used as a source; preferably an aqueous solution of calcium chloride or an aqueous solution of chloride. It is an aqueous solution of barium.
  • the cationic polymer of the cationic polymer layer of the polymer-coated crosslinked alginate gel fiber comprises polyamino acids, basic polysaccharides, basic polymers and the like. It is a cationic polymer selected from the group.
  • the cationic polymer of the cationic polymer layer of the polymer-coated crosslinked arginine gel fiber is preferably poly-L-ornithine (PLO), which is a polyamino acid.
  • Poly-D-ornithine PDO
  • poly-DL-ornithine poly-DL-ornithine
  • poly-D-lysine poly-L-lysine
  • PLA Poly-L-arginine
  • PDA Poly-D-Arginine
  • PHA Poly-DL-Arginine
  • PDHA Poly-L-Homoarginine
  • PDHA Poly-D-Homoarginine
  • Poly-DL-Homoarginine Poly-L-Histidine
  • PH Poly-D-arginine
  • PH Poly-DL-histidine
  • a cationic polymer selected from the group more preferably poly-L-arginine or poly-L-lysine; still more preferably. It is poly-L-ornithine.
  • the cationic polymer of the cationic polymer layer of the polymer-coated crosslinked alginate gel fiber is chitosan.
  • the cationic polymer of the cationic polymer layer of the polymer-coated crosslinked alginate gel fiber is polymethylene-CO-guanidine (PMCG), polyallylamine (PAA), and the like.
  • the outer diameter of the polymer-coated crosslinked alginate gel fiber is, for example, in the range of about 0.1 to about 2000 ⁇ m.
  • the polymer of the above embodiment is used.
  • a preferred embodiment of the crosslinked alginate gel in the core layer of the coated crosslinked alginate gel fiber may be optionally formed.
  • the polymer-coated cross-linked alginic acid gel fiber of the above-mentioned embodiment [1] or [1A] is preferably an alginic acid derivative represented by the formula (I) used for forming the cross-linked alginic acid gel.
  • 1-1-1] is selected from the alginic acid derivative or the preferred alginic acid derivative of the above aspect [1-1-5], and the alginic acid derivative represented by the formula (II) is in the above aspect [1-2-1].
  • the cationic polymer layer is a polymer-coated crosslinked alginate gel fiber selected from the cationic polymers according to the above aspect [1-15-1].
  • the polymer-coated cross-linked alginic acid gel fiber of the above-mentioned embodiment [1] or [1A] is preferably an alginic acid derivative represented by the formula (I) used for forming the cross-linked alginic acid gel.
  • 1-1-1] is selected from the alginic acid derivative or the preferred alginic acid derivative of the above aspect [1-1-5], and the alginic acid derivative represented by the formula (II) is in the above aspect [1-2-1].
  • the cationic polymer layer is a polymer-coated crosslinked alginate gel fiber selected from the cationic polymers according to the above aspect [1-15-1].
  • the polymer-coated cross-linked alginic acid gel fiber of the above-mentioned embodiment [1] or [1A] is more preferably the alginic acid derivative represented by the formula (I) used for forming the cross-linked alginic acid gel.
  • the alginic acid derivative selected from the alginic acid derivative described in [1-1-2] or the more preferable alginic acid derivative of the above embodiment [1-1-5] and represented by the formula (II) is the alginic acid derivative of the above embodiment [1-2-2]. ].
  • the alginic acid derivative described in [1-2-5] or the more preferable alginic acid derivative of the above aspect [1-2-5]; the antibody-producing cells are the cells according to the above aspects [1-3-2] to [1-3-3].
  • the cationic polymer layer is a polymer-coated crosslinked alginate gel fiber selected from the cationic polymers according to the above embodiments [1-15-2] to [1-15-4].
  • the polymer-coated cross-linked alginic acid gel fiber of the above-mentioned embodiment [1] or [1A] is more preferably the alginic acid derivative represented by the formula (I) used for forming the cross-linked alginic acid gel.
  • the alginic acid derivative selected from the alginic acid derivative described in [1-1-2] or the more preferable alginic acid derivative of the above embodiment [1-1-5] and represented by the formula (II) is the alginic acid derivative of the above embodiment [1-2-2]. ].
  • the polymer-coated cross-linked alginic acid gel fiber of the above-mentioned embodiment [1] or [1A] is more preferably the alginic acid derivative represented by the formula (I) used for forming the cross-linked alginic acid gel.
  • the alginic acid derivative represented by the formula (II) is selected from the alginic acid derivative described in [1-1-3], and the alginic acid derivative represented by the formula (II) is selected from the alginic acid derivative described in the above aspect [1-2-3]; the antibody-producing cell is described above. Selected from the cells according to embodiments [1-3-2] to [1-3-3]; the cationic polymer layer is described in embodiments [1-15-2] or [1-15-4].
  • the polymer-coated cross-linked alginic acid gel fiber of the above-mentioned embodiment [1] or [1A] is more preferably the alginic acid derivative represented by the formula (I) used for forming the cross-linked alginic acid gel.
  • the alginic acid derivative represented by the formula (II) is selected from the alginic acid derivative described in [1-1-3], and the alginic acid derivative represented by the formula (II) is selected from the alginic acid derivative described in the above aspect [1-2-3]; the antibody-producing cell is the embodiment.
  • the cationic polymer layer is the cation according to the above-mentioned embodiment [1-15-2] or [1-15-4].
  • a polymer-coated crosslinked alginate gel fiber selected from sex polymers.
  • the polymer-coated cross-linked alginic acid gel fiber of the above-mentioned embodiment [1] or [1A] is particularly preferably the alginic acid derivative represented by the formula (I) used for forming the cross-linked alginic acid gel.
  • the alginic acid derivative represented by the formula (II) is selected from the alginic acid derivative described in [1-1-4], and the alginic acid derivative represented by the formula (II) is selected from the alginic acid derivative described in the above aspect [1-2-4]; the antibody-producing cell is an antibody.
  • Producing CHO cells polymer-coated crosslinks in which the cationic polymer layer is selected from poly-L-ornithine, polyallylamine (PAA), polyethyleneimine, or polymethylene-CO-guanidine (PMCG). It is an alginate gel fiber.
  • PAA polyallylamine
  • PMCG polymethylene-CO-guanidine
  • the crosslinked alginate gel of the polymer-coated crosslinked alginate gel fiber is the above-mentioned embodiments [1-4] to [1-4]. It contains any of the components that can be added as described in -1].
  • cells capable of producing antibodies, physiologically active substances and the like, and chemically modified alginic acid derivatives represented by the formulas (I) and (II) used for forming the crosslinked alginic acid gel By combining each element of the cationic polymer (cationic polymer layer), a preferred embodiment of the method for producing a polymer-coated crosslinked alginate gel fiber can be arbitrarily formed.
  • the first aspect is as follows.
  • a multilayer polymer-coated crosslinked alginate gel fiber comprising a cationic polymer layer (cationic polymer) covering the core layer and an anionic polymer layer (anionic polymer) covering the cationic polymer layer.
  • the chemically modified alginic acid derivative represented by the formula (I) and the formula (II) is the same as that defined in the above aspect [1].
  • the aspect of the first C is as follows.
  • the chemically modified alginic acid derivative represented by the formula (I) and the formula (II) is the same as that defined in the above aspect [1].
  • the first aspect is as follows.
  • a multilayer polymer-coated crosslinked alginate gel fiber comprising a core layer, a cationic polymer layer disposed outside the core layer, and an anionic polymer layer disposed outside the cationic polymer layer, wherein the core layer.
  • Gel fiber The chemically modified alginic acid derivative represented by the formula (I) and the formula (II) is the same as that defined in the above aspect [1].
  • Akn - L1-1 of the chemically modified alginic acid derivative represented by the formula (I) is preferably the above-mentioned embodiment [1-1-1]. It is the same as the definition described in; more preferably the same as the definition described in the above aspect [1-1-2]; still more preferably described in the above aspect [1-1-3]. Same as the definition; particularly preferably the same as the definition described in the above aspect [1-1-4]; most preferably the following partial structural formula (in each formula, the right side of the broken line is not included): It is a group represented by.
  • the chemically modified alginic acid derivative ⁇ L2- represented by the formula ( II ) is preferably the above-mentioned embodiment [1-2-1].
  • the antibody and the physiologically active substance contained in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are, for example, various monoclonal antibodies such as human antibody, humanized antibody, chimeric antibody, mouse antibody or various modifications such as bispecific antibody thereof, low molecular weight antibody, sugar chain modified antibody and the like.
  • Examples include cells producing (type antibodies), cells producing physiologically active substances (enzymes, cytokines, hormones, blood coagulation factors, vaccines, etc.), cells capable of producing various useful substances useful as pharmaceutical raw materials, chemical raw materials, food raw materials, etc. ; Preferably antibody-producing cells or physiologically active substance-producing cells.
  • the antibody can be encapsulated in the core layer of a polymer-coated cross-linked alginate gel fiber or a multilayer polymer-coated cross-linked alginate gel fiber.
  • the antibody-producing cells are hybridomas (antibody-producing hybridomas) obtained from B cells that produce antibodies or cultured cells (antibody-producing recombinant cells) transformed with an antibody expression vector.
  • antibody-producing cells that can be encapsulated in the core layer of a polymer-coated crosslinked alginate gel fiber or a multilayer polymer-coated crosslinked alginate gel fiber.
  • the animal cell used as a host is a CHO cell, a CHO cell substrain (CHO-K1 cell, CHO-DG44 cell, CHO-DXB11 cell, or CHO cells transformed to modify sugar chains, etc.), COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, PERC6 cells, YB2 / 0 cells, YE2 / 0 cells, 1R983F cells, Namalwa cells, Will.
  • CHO cell a CHO cell substrain (CHO-K1 cell, CHO-DG44 cell, CHO-DXB11 cell, or CHO cells transformed to modify sugar chains, etc.)
  • COS cells COS cells
  • Sp2 / 0 cells NS0 cells
  • SP2 cells SP2 cells
  • PERC6 cells YB2 / 0 cells
  • YE2 / 0 cells YE2 / 0 cells
  • 1R983F cells Namalwa cells, Will.
  • -2 cells Jurkat cells, Vero cells, Molt-4 cells, HEK293 cells, BHK cells, HT-1080 cells, KGH6 cells, P3X63Ag8.653 cells, C127 cells, JC cells, LA7 cells, ZR-45-30 cells, A cell selected from hTERT cells, NM2C5 cells, or UACC-812 cells.
  • the animal cells used as a host are preferably CHO cells, CHO cell substrains, COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells. , PERC6 cells, HEK293 cells, BHK cells, HT-1080 cells, or C127 cells; more preferably, CHO cells, CHO cell substrains, Sp2 / 0 cells, NS0 cells, HEK293 cells, or A cell selected from BHK cells; more preferably a CHO cell or a CHO cell substrain.
  • the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are ,
  • the host cell is a cell selected from CHO cells, CHO cell substrains, Sp2 / 0 cells, or NS0 cells; more preferably, CHO cells or CHO cell substrains.
  • the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated crosslinked alginate gel fiber or the multilayer polymer-coated crosslinked alginate gel fiber are , Biopharmacy or cells producing antibodies used as biopharmacy raw materials.
  • the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are ⁇ -CD3 ⁇ , Brentuximab, Inotsumab, Polatsuzumab, Enholtumab, Sashitsuzumab, Verantamab, Roncastuzumab, Chisotumab, Datopotamab, Patritumab and other antibody-producing cells; Cells; cells that produce low molecular weight antibodies consisting of antibody fragments such as ranibizmab, idalcizumab, brinatsumomab, brolcizumab, absiximab, couplersizumab, and sertrizumab.
  • the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are , Antibody-producing animal cells, preferably antibody-producing CHO cells, antibody-producing Sp2 / 0 cells or antibody-producing NS0 cells, and more preferably antibody-producing CHO cells.
  • the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber are Preferably, there are antibody-producing CHO cells whose host cells are CHO cells, such as muromonab-CD3-producing CHO cells, trussumab-producing CHO cells, rituximab-producing CHO cells, paribizmab-producing CHO cells, infriximab-producing CHO cells, and basiliximab-producing CHO cells.
  • CHO cells such as muromonab-CD3-producing CHO cells, trussumab-producing CHO cells, rituximab-producing CHO cells, paribizmab-producing CHO cells, infriximab-producing CHO cells, and basiliximab-producing CHO cells.
  • tosirizumab-producing CHO cells gemtuzumab-producing CHO cells, bebashizumab-producing CHO cells, ibritsumomab-producing CHO cells, adalimumab-producing CHO cells, setuximab-producing CHO cells, ranibizmab-producing CHO cells, omalizumab-producing CHO cells, ecrizumab-producing CHO cells, ecrizumab-producing CHO cells Cells, Ustekinumab-producing CHO cells, Golimumab-producing CHO cells, Kanakinumab-producing CHO cells, Denosumab-producing CHO cells, Mogamurizumab-producing CHO cells, Celtrizumab-producing CHO cells, Ofatumumab-producing CHO cells, Peltzzumab-producing CHO cells, Kunststoffuccimab-producing CHO cells, Producing CHO cells, nibolumab-producing CHO cells, alemtu
  • the cells are the same as the physiologically active substance-producing cells described in the above aspect [1-3-4].
  • the cell is a cell selected from the group consisting of insulin secreting cells, islets, islet cells, or MIN6 cells derived from pancreatic ⁇ cells.
  • the cells are cultured cells (physiologically active substance-producing gene recombinant cells) transformed with a physiologically active substance expression vector.
  • production of a physiologically active substance that can be encapsulated in the core layer of a polymer-coated crosslinked alginate gel fiber or a multilayer polymer-coated crosslinked alginate gel fiber.
  • the cell is a recombinant animal cell that produces a physiologically active substance.
  • the animal cells used as a host include CHO cells, CHO cell substrains, COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, or Cells selected from PERC6 cells, HEK293 cells, BHK cells, HT-1080 cells, or C127 cells; preferably CHO cells, CHO cell substrains, Sp2 / 0 cells, and NS0 cells, HEK293 cells, or BHK.
  • the cell is preferably a cell whose host cell is selected from CHO cells, CHO cell substrains, HEK293 cells, or BHK cells; more preferably CHO cells, or CHO cell substrains.
  • a cell is a cell that produces a biopharmaceutical or a physiologically active substance used as a raw material for a biopharmaceutical.
  • the cells are interferonase, interferonase, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, interferon, inter
  • Cells Protein-producing cells; insulin, insulin lispro, insulin aspart, insulin glargine, insulin detemil, insulin glulysine, insulin degludec, somatropin, somapcitan, mechacermin, carperitide, bosolitide, glucagon, holitropin, coriogonadotropin, duraglutide, liglutide, , Teriparatide, cells producing hormones such as metrereptin; cells producing interferon such as interferon alpha-2a, interferon alpha-2b, interferon beta-1a, interferon beta-1b, interferon gamma-1a; cells producing epoetin, darbepoetin, lomiprostim, etc.
  • Cells that produce hematopoietic factors include cells that produce hematopoietic factors; cells that produce cytokines and their receptors such as Philgrastim, Lenograstim, Teseloykin, Trafermin, Belfermin, Etanelcept, Afribelcept, Deniloukin diphthitox; Cell surface antigens such as Avatacept, A cell selected from cells that produce cell surface receptors and their ligands.
  • the cell is a bioactive substance-producing animal cell, preferably a bioactive substance-producing CHO cell, a bioactive substance-producing HEK293 cell, or a bioactive substance-producing BHK cell, and more preferably a bioactive substance-producing CHO cell. ..
  • the cell is preferably a physiologically active substance-producing cell CHO cell whose host cell is a CHO cell, for example, an alteplase-producing CHO cell, an alglucosidase-producing CHO cell, a rulioctocog-producing CHO cell, a duraglutide-producing CHO cell, an interferon beta-. 1a-producing CHO cells, darbepoetin-producing CHO cells, etanercept-producing CHO cells, afribercept-producing CHO cells, or avatarcept-producing CHO cells.
  • the components that can be additionally contained in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber are the same as the components described in the above-mentioned aspect [1-4].
  • the preferred component is the same as the component described in the above aspect [1-4-1].
  • the concentration of the alginic acid solution that can be additionally contained in the core layer of the multilayer polymer-coated crosslinked alginic acid gel fiber, or the concentration of the alginic acid solution used for forming the alginic acid gel. Is the same as the concentration range described in the above aspect [1-10-4].
  • the crosslinked alginate gel contained in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber contains an alginate solution or an alginate gel formed from the alginic acid solution.
  • the total concentration of the mixed solution containing the chemically modified arginic acid derivatives represented by the formulas (I) and (II) used for forming the core layer and the concentration of the arginic acid solution is preferably about 0.5 to about. 2.0% by weight; more preferably a concentration selected from about 1.0% by weight, about 1.5% by weight and about 2.0% by weight; even more preferably at about 1.5% by weight. be.
  • the crosslinked alginic acid gel contained in the core layer of the multilayer polymer-coated crosslinked alginic acid gel fiber contains an alginic acid solution or an alginic acid gel formed from the alginic acid solution. If so, the concentration of the solution of the chemically modified alginic acid derivative represented by the formula (I) used for forming the core layer (C1A (% by weight)), the concentration of the solution of the chemically modified alginic acid derivative represented by the formula (II) (C1N).
  • the chemically modified alginic acid represented by the formula (I) used for forming the crosslinked alginic acid gel contained in the core layer of the multilayer polymer-coated crosslinked alginic acid gel fiber used for forming the crosslinked alginic acid gel contained in the core layer of the multilayer polymer-coated crosslinked alginic acid gel fiber.
  • the crosslinked alginic acid gel contained in the core layer of the multilayer polymer-coated crosslinked alginic acid gel fiber contains an alginic acid solution or an alginic acid gel formed from the alginic acid solution. If so, the solution of each solution in a mixed solution of a chemically modified alginic acid derivative represented by the formula (I), a chemically modified alginic acid derivative solution represented by the formula (II), and an alginic acid solution used for forming the core layer.
  • the crosslinked alginate gel contained in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber is the formula (III-L) described in the above aspect [1-12]. ) (The definition in the formula is the same as the definition in the above-mentioned embodiment [1-12]).
  • the crosslinked alginate gel contained in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber is the formula (III-L) according to the embodiment [1-12A].
  • Each definition in formula (III-L) comprises a group-mediated chemical cross-linking represented by the above-mentioned embodiment [1-12A].
  • the crosslinked alginate gel contained in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber is the formula (III-L) [formula] of the embodiment [1-12].
  • each definition is the same as the definition of the above-mentioned embodiment [1-12]] or each of the above-mentioned embodiments [1B-12A] in the formulas (III-L) [formula (III-L)].
  • the definition is the same as the definition of aspect [1B-12A]] and includes chemical cross-linking via a group and ionic cross-linking via a divalent metal ion.
  • the divalent metal ion used for forming an ionic crosslink in the crosslinked alginate gel contained in the core layer is the divalent metal ion described in the above aspect [1-13A]. It is the same as the valence metal ion.
  • the cationic polymer of the cationic polymer layer of the multilayer polymer-coated crosslinked alginate gel fiber is the cation described in the above-mentioned embodiment [1-15-1]. Same as sex polymer.
  • the cationic polymer of the cationic polymer layer of the multilayer polymer coated crosslinked alginate gel fiber is chitosan.
  • the cationic polymer of the multilayer polymer-coated crosslinked alginate gel fiber is the same as the cationic polymer described in the above-mentioned embodiment [1-15-4]. be.
  • the anionic polymer layer of the multilayer polymer-coated crosslinked alginate gel fiber is an anionic polysaccharide, a sulfated polysaccharide, a synthetic polymer, an anionic polyamino acid, and the like.
  • An anionic polymer selected from the group consisting of chemically modified products, crosslinked products thereof, mixtures thereof, and the like.
  • the anionic polymer layer of the multilayer polymer-coated crosslinked alginic acid gel fiber is, for example, alginic acid, hyaluronic acid, capolic galacturonic acid, laginan, succinoglucan, and the like.
  • the anionic polymer layer of the multilayer polymer coated crosslinked alginic acid gel fiber is alginic acid.
  • the anionic polymer layer of the multilayer polymer-coated crosslinked alginate gel fiber is the formula (I) of the above-mentioned embodiment [1] or [1-1-5].
  • the anionic polymer layer of the multilayer polymer-coated crosslinked alginic acid gel fiber is alginic acid, and the formula (1B-16-4) of the above-mentioned embodiment [1] or [1-1-5] is used.
  • It consists of a crosslinked product formed from a chemically modified alginic acid derivative represented by the formula (I) and a chemically modified alginic acid derivative represented by the formula (II) of the above embodiment [1] or [1-2-5], and a mixture thereof.
  • An anionic polymer selected from the group.
  • the anionic polymer layer of the multilayer polymer-coated crosslinked alginate gel fiber is, for example, chondroitin sulfate, dextran sulfate, heparan sulfate, dermatan sulfate, fucoidan, keratane sulfate. , Or an anionic polymer selected from the group consisting of sulfated polysaccharides such as heparan, chemically modified products thereof, crosslinked products thereof, mixtures thereof, and the like.
  • the anionic polymer layer of the multilayer polymer-coated crosslinked alginate gel fiber is synthesized from, for example, acrylic acid, methacrylic acid, ethyl acrylic acid, polystyrene sulfonic acid and the like.
  • An anionic polymer selected from the group consisting of polymers, chemically modified products thereof, crosslinked products thereof, mixtures thereof, and the like.
  • the anionic polymer layer of the multilayer polymer-coated crosslinked alginate gel fiber is, for example, anionic polyamino acids such as polyglutamic acid and polyaspartic acid, and their chemistry.
  • the outer diameter of the multilayer polymer-coated crosslinked alginate gel fiber is, for example, in the range of about 0.1 to about 2000 ⁇ m.
  • the multilayer of the above embodiment is used.
  • a preferred embodiment of the crosslinked alginate gel in the core layer of the polymer coated crosslinked alginate gel fiber may be optionally formed.
  • the multilayer polymer-coated cross-linked alginic acid gel fiber of the above-mentioned embodiments [1B] to [1D] is preferably an alginic acid derivative represented by the formula (I) used for forming the cross-linked alginic acid gel.
  • the alginic acid derivative selected from the alginic acid derivative according to [1-1-1] or the preferred alginic acid derivative according to the above aspect [1-1-5] and represented by the formula (II) is the alginic acid derivative according to the above aspect [1-2-1].
  • the alginic acid derivative according to the above or the preferred alginic acid derivative according to the above embodiment [1-2-5]; the antibody-producing cells are selected from the cells according to the above embodiments [1B-3-1] to [1B-3-9]. Selected; the cationic polymer layer is selected from the cationic polymer according to the above embodiment [1-15-1]; the anionic polymer layer is selected from the above embodiments [1B-16] to [1B-16-7].
  • a multilayer polymer coated crosslinked alginate gel fiber selected from the anionic polymers described.
  • the multilayer polymer-coated cross-linked alginic acid gel fiber of the above-described embodiments [1B] to [1D] is more preferably the alginic acid derivative represented by the formula (I) used for forming the cross-linked alginic acid gel.
  • the alginic acid derivative selected from the alginic acid derivative according to the embodiment [1-1-2] or the more preferable alginic acid derivative according to the above embodiment [1-1-5] and represented by the formula (II) is the alginic acid derivative according to the embodiment [1-2-2].
  • -1] is a multilayer polymer-coated crosslinked alginate gel fiber selected from the anionic polymers described in -1].
  • the multilayer polymer-coated cross-linked alginic acid gel fiber of the above-described embodiments [1B] to [1D] is more preferably the alginic acid derivative represented by the formula (I) used for forming the cross-linked alginic acid gel.
  • the alginic acid derivative represented by the formula (II) is selected from the alginic acid derivative according to the embodiment [1-1-3], and the alginic acid derivative represented by the formula (II) is selected from the alginic acid derivative according to the embodiment [1-2-3]; Selected from the cells according to the embodiments [1B-3-3] to [1B-3-9]; the cationic polymer layer is described in the embodiments [1-15-2] or [1-15-4].
  • the anionic polymer layer is selected from the anionic polymers according to the above embodiments [1B-16-2] to [1B-16-4-4]; the multilayer polymer coated crosslinked alginate gel. It is a fiber.
  • the multilayer polymer-coated cross-linked alginic acid gel fiber of the above-described embodiments [1B] to [1D] is particularly preferably the alginic acid derivative represented by the formula (I) used for forming the cross-linked alginic acid gel.
  • the alginic acid derivative represented by the formula (II) is selected from the alginic acid derivative described in the embodiment [1-1-4], and the alginic acid derivative represented by the formula (II) is selected from the alginic acid derivative described in the above aspect [1-2-4]; Selected from the cells according to the embodiments [1B-3-6] to [1B-3-9]; the cationic polymer layer is poly-L-alginic acid, polyallylamine (PAA), polyethyleneimine, or polymethylene-CO. -Selected from guanidine (PMCG); the anionic polymer layer is alginic acid, formula (I) according to the above embodiments [1-1-1] to [1-1-4], [1-2-5].
  • -2-4 a multilayer polymer-coated cross-alginic acid gel fiber selected from cross-linked bodies formed from the alginic acid derivative represented by the formula (II) described in [1-2-4].
  • the multilayer polymer-coated crosslinked alginic acid gel fiber according to the above-described embodiments [1B] to [1D] is most preferably the alginic acid derivative represented by the formula (I) used for forming the crosslinked alginic acid gel.
  • the following partial structural formula as Akn-L 1- (in each formula, the right side of the broken line is not included): It has a group represented by; the alginic acid derivative represented by the formula ( II ) is ⁇ L2-.
  • the antibody-producing cells are selected from the cells according to the above aspect [1B-3-5], preferably the antibody-producing cells are antibody-producing CHO cells; the cationic polymer layer is , Poly-L-ornithine, polyallylamine (PAA), polyethyleneimine, or polymethylene-CO-guanidine (PMCG);
  • the anionic polymer layer is alginic acid, Akn-L 1- as the following partial structural formula (in each formula, the right side of the broken line is not included):
  • the alginic acid derivative represented by the formula (I), ⁇ L2- As the alginic acid derivative represented by the formula (I), ⁇ L2- , the following partial structural formula (in each formula, the outside of the broken line at both ends is not included):
  • An alginic acid derivative represented by the formula (II) and an alginic acid represented by the formula (I) having the partial structural formula (ALK-1a-3a) or (ALK-2-3) as Akn - L1-.
  • a multilayer polymer coated cross-linked alginate gel fiber selected from cross-linked bodies.
  • the crosslinked alginate gel of the multilayer polymer coated crosslinked alginate gel fiber is the above-mentioned embodiments [1-4] to [1-].
  • 4-1] contains any of the addable components described in.
  • a preferred embodiment of the method for producing a multilayer polymer-coated crosslinked alginate gel fiber can be arbitrarily formed.
  • the second aspect is as follows. Performing a cross-linking reaction using a cell capable of producing an antibody, a physiologically active substance, etc., a chemically modified alginic acid derivative represented by the formula (I) and a chemically modified alginic acid derivative represented by the formula (II) according to the above embodiment [1].
  • a method for producing a polymer-coated cross-linked alginic acid gel fiber formed by coating a core layer containing the cross-linked alginic acid gel obtained in the above with a cationic polymer.
  • Step (1) A divalent metal containing a cell capable of producing an antibody, a physiologically active substance, or the like, and a chemically modified alginic acid derivative represented by the formulas (I) and (II) described in the above embodiment [1].
  • Step (2) A cation by contacting a crosslinked alginate gel fiber (CLA) containing cells capable of producing the antibody, physiologically active substance, etc. obtained in the step (1) in a core layer with a solution containing a cationic polymer.
  • the production method comprises the steps of obtaining a polymer-coated crosslinked alginate gel fiber (CFB) coated with a sex polymer layer.
  • the polymer-coated crosslinked alginate gel fiber according to the above aspect [2] is the polymer-coated crosslinked alginate gel fiber according to any one of the above aspects ([1] to [1-17-5]).
  • the cells capable of producing the antibody, the physiologically active substance, etc. used for producing the polymer-coated crosslinked alginate gel fiber are the cells described in the above aspects [1-3] to [1-3-5]. It is the same as the cell capable of producing the antibody, the physiologically active substance, etc. according to any one of the above items.
  • cells capable of producing an antibody, a physiologically active substance, etc. used for producing a polymer-coated crosslinked alginate gel fiber are the cells described in the above aspects [1B-3] to [1B-3-]. It is the same as the cell capable of producing the antibody, physiologically active substance, etc. according to any one of 19].
  • the weight average molecular weight measured by the gel filtration chromatography method of the chemically modified alginic acid derivative represented by the formula (I) used for producing the polymer-coated crosslinked alginic acid gel fiber is, for example, It ranges from about 100,000 Da to about 3,000,000 Da; preferably in the range of about 300,000 Da to about 2,500,000 Da, more preferably in the range of about 500,000 Da to about 2,000,000 Da. Is.
  • the weight average molecular weight measured by the gel filtration chromatography method of the chemically modified alginic acid derivative represented by the formula (II) used for producing the polymer-coated crosslinked alginic acid gel fiber is, for example, It ranges from about 100,000 Da to about 3,000,000 Da; preferably in the range of about 300,000 Da to about 2,500,000 Da, more preferably in the range of about 500,000 Da to about 2,000,000 Da. Is.
  • L1 ⁇ is the same as the definition in the above embodiments [ 1 ] to [1-1-4]), for example, the introduction rate is in the range of about 0.1 to about 30 mol%; preferably about. It is in the range of 0.3 to about 20 mol%; more preferably in the range of about 0.5 to about 10 mol%.
  • the reactive group of the chemically modified alginate derivative represented by the formula (II) used in the production of the polymer-coated crosslinked alginate gel fiber N3- L2-NH 2 groups ( -).
  • L2- has the same definition as in the above embodiments [ 1 ], [1-2-1] to [1-2-4]), and the introduction rate is, for example, about 0.1 to about 30 mol%. It is in the range; preferably in the range of about 0.3 to about 20 mol%; more preferably in the range of about 0.5 to about 15 mol%.
  • it is a component selected from the group consisting of an alginic acid solution, a medium, a culture solution, a collagen solution, methyl cellulose, a sucrose solution, or a mixture thereof; preferably, an alginic acid solution, a medium, a culture solution, or a mixture thereof, etc. It is a component selected from the group consisting of.
  • the weight average molecular weight of alginic acid used in (eg, sodium alginate, etc.) measured by gel filtration chromatography (GPC method) is, for example, in the range of about 150,000 Da to about 2.5 million Da; preferably. It ranges from about 300,000 Da to about 2,000,000 Da, more preferably from about 700,000 Da to about 1,500,000 Da.
  • the weight average molecular weight of alginic acid (eg, sodium alginate, etc.) used to prepare an alginic acid solution that can be added to the solution as measured by a gel filtration chromatography method (GPC method) is, for example, about 150,000 Da to about 2,500, It is in the range of 000 Da; preferably in the range of about 300,000 Da to about 2.5 million Da; more preferably in the range of about 700,000 Da to about 1.400,000 Da, about 800,000 Da to about 1.5 million Da. , About 1,400,000 to about 2,000,000 Da, or about 1,500,000 to about 2,500,000.
  • the concentration of the solution of the chemically modified alginate derivative represented by the formula (I) used for producing the polymer-coated crosslinked alginate gel fiber is, for example, about 0.01 to about 1. It is in the range of 5% by weight; preferably in the range of about 0.05 to about 1.0% by weight; more preferably in the range of about 0.08 to about 0.75% by weight.
  • the concentration of the solution of the chemically modified alginate derivative represented by the formula (II) used for producing the polymer-coated crosslinked alginate gel fiber is, for example, about 0.01 to about 1. It is in the range of 5% by weight; preferably in the range of about 0.05 to about 1.0% by weight; more preferably in the range of about 0.08 to about 0.75% by weight.
  • a mixed solution of a chemically modified arginic acid derivative represented by the formula (I) and a chemically modified arginic acid derivative represented by the formula (II) used for producing a polymer-coated crosslinked arginic acid gel fiber is, for example, in the range of about 0.02 to about 2.0% by weight; preferably in the range of about 0.1 to about 2.0% by weight; more preferably about 0.15 to about 1%. It is in the range of 5.5% by weight.
  • the concentration of the alginic acid solution that can be added to the mixed solution containing the cells capable of producing an antibody, a physiologically active substance and the like and the chemically modified alginic acid derivatives represented by the formulas (I) and (II). Is, for example, in the range of 0 to about 1.98% by weight; preferably in the range of 0 to about 1.8% by weight; more preferably in the range of 0 to about 1.7% by weight.
  • the alginic acid solution is added to a mixed solution containing cells capable of producing an antibody, a physiologically active substance, etc. and a chemically modified alginic acid derivative represented by the formulas (I) and (II).
  • the total concentration of the mixed solution containing the chemically modified alginic acid derivative represented by the formula (I) and the formula (II) and the concentration of the alginic acid solution is preferably about 0.5 to about 2.0% by weight. Yes; more preferably, the concentration is selected from about 1.0% by weight, about 1.5% by weight and about 2.0% by weight.
  • the alginic acid solution is added to a mixed solution containing cells capable of producing an antibody, a physiologically active substance, etc. and a chemically modified alginic acid derivative represented by the formulas (I) and (II).
  • the combination of the concentration (C1 (% by weight)) of the mixed solution containing the chemically modified alginic acid derivative represented by the formulas (I) and (II) and the concentration of the alginic acid solution (C2 (% by weight)) is preferable.
  • (C1: C2) (about 0.2: about 1.3), (about 0.5: about 1.0), (about 1.0: about 0.5), (about 1.5: It is a combination selected from the group consisting of 0), (about 0.66: about 1.34), and (about 0.34: about 0.66).
  • the alginic acid solution is added to a mixed solution containing cells capable of producing an antibody, a physiologically active substance, etc. and a chemically modified alginic acid derivative represented by the formulas (I) and (II).
  • cells capable of producing an antibody, a physiologically active substance, etc. used for forming a core layer of a polymer-coated crosslinked alginate gel fiber and represented by formulas (I) and (II).
  • the alginic acid solution is added to the mixed solution containing the chemically modified alginic acid derivative
  • the volume (v1) of the chemically modified alginic acid derivative represented by the formula (I) in the mixed solution to which the alginic acid solution is added is represented by the formula (II).
  • a mixed solution containing a cell capable of producing an antibody, a physiologically active substance, etc. and a chemically modified alginic acid derivative represented by the formulas (I) and (II) is injected into the solution.
  • the divalent metal ion contained is a divalent metal ion selected from the group of calcium ion, magnesium ion, barium ion, strontium ion, zinc ion and the like; preferably calcium ion, barium ion or strontium ion. More preferably, it is calcium ion or barium ion.
  • the solution for injecting a mixed solution containing cells capable of producing an antibody, a physiologically active substance, etc. and a chemically modified alginic acid derivative represented by the formulas (I) and (II) is a solution.
  • the concentration of the divalent metal ion is, for example, in the range of about 1 mM to about 1 M, or about 10 to about 500 mM; preferably. It is about 10 to about 100 mM.
  • the mixed solution containing the cells capable of producing an antibody, a physiologically active substance, etc. and the chemically modified alginic acid derivative represented by the formulas (I) and (II) is, for example, FIG.
  • the mixed solution can be introduced from the introduction port 1 of the device XX and ejected from the discharge port 2 of the device XX.
  • the mixed solution containing the cells capable of producing an antibody, a physiologically active substance, etc. and the chemically modified alginic acid derivative represented by the formulas (I) and (II) For example, the ejection can be performed from the discharge port 2 of the apparatus XX using an extrusion cylinder YY or the like as shown in FIG.
  • an injection cylinder can be used as a combination of the apparatus XX and the extruder YY.
  • a glass or plastic syringe barrel can be used as the syringe barrel.
  • [2-18] In the above-mentioned embodiments [2], [2-16-1] and [2-16-2], cells capable of producing an antibody, a physiologically active substance and the like, and represented by the formulas (I) and (II).
  • the injection rate (flow velocity) of the mixed solution containing the chemically modified alginic acid derivative is, for example, in the range of about 100 to about 10000 ⁇ L / min.
  • the solution containing the cationic polymer to be contacted with the crosslinked alginate gel fiber (CLA) containing cells capable of producing an antibody, a physiologically active substance, etc. is a polyamino acid (basic amino acid).
  • Polymer a basic polysaccharide, a basic polymer, a solution containing a cationic polymer selected from the group consisting of salts thereof and the like.
  • the solution containing a cationic polymer to be contacted with a crosslinked ornithine gel fiber (CLA) containing cells capable of producing an antibody, a physiologically active substance, or the like is preferably a polyamino acid.
  • the solution containing a cationic polymer to be contacted with a crosslinked alginate gel fiber (CLA) containing cells capable of producing an antibody, a physiologically active substance, etc. is, for example, a basic polysaccharide.
  • the solution containing the cationic polymer to be contacted with the crosslinked alginate gel fiber (CLA) containing cells capable of producing an antibody, a physiologically active substance, etc. is, for example, a basic polymer.
  • a basic polymer Select from the group consisting of certain polymethylene-CO-guanidine (PMCG), polyallylamine (PAA), polyvinylamine (PVA), polyethyleneimine, allylamine-diallylamine copolymers, allylamine-maleic acid copolymers, and salts thereof. It is a solution containing a cationic polymer to be used.
  • the solution containing the cationic polymer is polyallylamine (PAA), polyethyleneimine, polymethylene-CO-guanidine (PMCG) and them.
  • PAA polyallylamine
  • PMCG polymethylene-CO-guanidine
  • the solution containing a cationic polymer to be contacted with a crosslinked alginate gel fiber (CLA) containing cells capable of producing an antibody, a physiologically active substance, etc. is a calcium chloride solution, a buffer solution, or the like.
  • the temperature during production of the polymer-coated crosslinked alginate gel fiber is, for example, in the range of about 4 to about 37 ° C.
  • a preferred embodiment of the method for producing a polymer-coated crosslinked alginate gel fiber can be arbitrarily formed.
  • a cross-linking reaction is carried out using a cell capable of producing an antibody, a physiologically active substance or the like, a chemically modified arginic acid derivative represented by the formula (I) and a chemically modified arginic acid derivative represented by the formula (II) according to the above embodiment [1].
  • a method for producing a multilayer polymer-coated crosslinked alginate gel fiber formed by sequentially coating a core layer containing a crosslinked alginate gel obtained in the above step with a cationic polymer and an anionic polymer.
  • Step (1) A mixture containing cells capable of producing an antibody, a physiologically active substance, etc., and a chemically modified alginic acid derivative represented by the formula (I) and a chemically modified alginic acid derivative represented by the formula (II) according to the above embodiment [1].
  • Step (2) A cross-linked alginate gel fiber (CLA) containing a cell capable of producing an antibody, a physiologically active substance, etc.
  • a step of obtaining a polymer-coated crosslinked alginate gel fiber (CFB) coated with a sex polymer layer Step (3): The polymer-coated crosslinked alginate gel fiber (CFB) obtained in step (2) is brought into contact with a solution containing an anionic polymer to obtain a multilayer polymer-coated crosslinked alginate gel coated with an anionic polymer layer.
  • the process of obtaining fiber (ACFB), It is a manufacturing method characterized by containing.
  • the multilayer polymer-coated crosslinked alginate gel fiber according to the embodiment [2B] is the multilayer polymer-coated crosslinked alginate gel fiber according to any one of the above embodiments ([1B] to [1B-18-6]).
  • cells capable of producing an antibody, a physiologically active substance, etc. used for producing a fiber are the cells of the above aspect [1-3] to [1-3-5], [1B]. -3] It is the same as the cell capable of producing the antibody, physiologically active substance, etc. according to any one of [1B-3-19].
  • the introduction rate of the two units (Akn-L 1 -is the same as the definition in the above-mentioned embodiment [1], [1B-1-1] or [1B-1-2]) is the introduction rate of the above-mentioned embodiment [2-4]. ] Is the same as the range of introduction rate described in.
  • the weight average molecular weight of alginic acid (for example, sodium alginate, etc.) used in the preparation as measured by the gel filtration chromatography method (GPC method) is the same as the molecular weight described in the above aspect [2-7A].
  • a cell capable of producing an antibody, a physiologically active substance, etc. and a chemically modified alginic acid derivative represented by the formulas (I) and (II) are included in the fiber manufacturing step (1).
  • the concentration of the alginic acid solution that can be added to the mixed solution is the same as the concentration range described in the above aspect [2-11].
  • the alginic acid solution is added to the mixed solution containing, the total concentration of the mixed solution containing the chemically modified alginic acid derivative represented by the formulas (I) and (II) and the concentration of the alginic acid solution is the above-mentioned embodiment [2-. It is the same as the concentration range described in 11-1]; more preferably, it is about 1.5% by weight.
  • the combination of% by weight)) is the same as the concentration range described in the above aspect [2-11-2].
  • the concentration (C1A (% by weight)) of the solution of the chemically modified alginic acid derivative represented by the formula (I) is added to the mixed solution containing the above, the concentration (C1A (% by weight)) of the solution of the chemically modified alginic acid derivative represented by the formula (I), and the solution of the chemically modified alginic acid derivative represented by the formula (II).
  • the combination of the concentration (C1N (% by weight)) and the concentration of the alginic acid solution (C2 (% by weight)) is the same as the range of the concentration described in the above aspect [2-11-3].
  • (v1: v2) (7.5: 7.5).
  • the alginic acid solution is added to the mixed solution containing, the volume (v1) of the chemically modified alginic acid derivative represented by the formula (I) and the chemically modified alginic acid derivative represented by the formula (II) in the mixed solution to which the alginic acid solution is added.
  • the volume ratio of the volume of the solution (v2) and the volume of the alginic acid solution (v3) is the same as the combination described in the above aspect [2-12-2].
  • a cell capable of producing an antibody, a physiologically active substance, etc. and a chemically modified alginic acid derivative represented by the formulas (I) and (II) are included.
  • the divalent metal ion contained in the solution for injecting the mixed solution is the same as the divalent metal ion described in the above aspect [2-13].
  • the concentration of the divalent metal ion used in the fiber manufacturing step (1) is, for example, in the range of about 1 mM to about 1 M, or about 10 to about. It is in the range of 500 mM; preferably about 10 to about 100 mM.
  • the mixed solution containing the cells capable of producing an antibody, a physiologically active substance, etc. and the chemically modified alginic acid derivative represented by the formulas (I) and (II) is, for example, FIG.
  • the mixed solution can be introduced from the introduction port 1 of the device XX and ejected from the discharge port 2 of the device XX.
  • the mixed solution containing the cells capable of producing an antibody, a physiologically active substance, etc. and the chemically modified alginic acid derivatives represented by the formulas (I) and (II) For example, the ejection can be performed from the discharge port 2 of the apparatus XX using an extrusion cylinder YY or the like as shown in FIG.
  • an injection cylinder can be used as a combination of the apparatus XX and the extrusion cylinder YY.
  • a glass or plastic syringe barrel can be used as the syringe barrel.
  • [2B-18] In the above-mentioned embodiments [2B], [2B-16-1] and [2B-16-2], cells capable of producing an antibody, a physiologically active substance and the like, and represented by formulas (I) and (II).
  • the injection rate (flow velocity) of the mixed solution containing the chemically modified alginic acid derivative is, for example, in the range of about 100 to about 10000 ⁇ L / min.
  • the fiber manufacturing step (2) comprises a cationic polymer to be contacted with a crosslinked alginate gel fiber (CLA) containing cells capable of producing an antibody, a physiologically active substance, or the like.
  • CLA crosslinked alginate gel fiber
  • a crosslinked alginate gel fiber (CLA) containing cells capable of producing an antibody, a physiologically active substance, or the like is contacted, which is preferable and more preferable.
  • the solutions containing the more preferred cationic polymers are the same as the solutions described in the above-mentioned embodiment [2-19-2], respectively.
  • the fiber manufacturing step (2) comprises a cationic polymer to be contacted with a crosslinked alginate gel fiber (CLA) containing cells capable of producing an antibody, a physiologically active substance, or the like.
  • the solution is, for example, a solution containing a cationic polymer selected from the group consisting of the basic polysaccharide chitosan or a salt thereof.
  • the fiber manufacturing step (2) comprises a cationic polymer to be contacted with a crosslinked alginate gel fiber (CLA) containing cells capable of producing an antibody, a physiologically active substance, or the like.
  • CLA crosslinked alginate gel fiber
  • the solution containing a cationic polymer to be contacted with a crosslinked alginate gel fiber (CLA) containing cells capable of producing an antibody, a physiologically active substance, or the like is used.
  • CLA crosslinked alginate gel fiber
  • Calcium chloride solution, buffer solution and the like can be contained.
  • the temperature during production of the multilayer polymer coated crosslinked alginate gel fiber is, for example, in the range of about 4 to about 37 ° C.
  • the solution containing the anionic polymer to be contacted with the polymer-coated crosslinked alginate gel fiber (CFB) is an anionic polysaccharide or a sulfated polysaccharide.
  • the solution containing the anionic polymer to be contacted with the polymer-coated crosslinked alginate gel fiber (CFB) in the fiber manufacturing step (3) is alginic acid, according to the above aspect [1].
  • an anionic polymer selected from the group consisting of a crosslinked product formed from a chemically modified alginic acid derivative and / or a chemically modified alginic acid derivative represented by the formula (II), salts thereof, and a mixture thereof.
  • the solution containing the anionic polymer to be contacted with the polymer-coated crosslinked alginate gel fiber is, for example, alginic acid, hyaluronic acid, capoli.
  • Anionic polysaccharides such as galacturonic acid, laginan, succinoglucan, gum arabic, xanthan gum, alginic acid, pectin, pectinic acid, carboxymethyl cellulose, agar, their chemically modified products, their bridges, their salts, and theirs.
  • a solution containing an anionic polymer selected from the group consisting of mixtures.
  • the solution containing the anionic polymer to be contacted with the polymer-coated crosslinked arginate gel fiber (CFB) is, for example, chondroitin sulfate, dextran sulfate, heparan.
  • cationic polymers selected from the group consisting of sulfated polysaccharides such as sulfuric acid, dermatan sulfate, fucoidan, keratane sulfate, or heparin, their chemical modifications, crosslinked products thereof, salts thereof, and mixtures thereof. It is a solution.
  • the solution containing the anionic polymer to be contacted with the polymer-coated crosslinked alginate gel fiber (CFB) is, for example, acrylic acid, methacrylic acid, ethyl.
  • the solution containing the anionic polymer to be contacted with the polymer-coated crosslinked alginate gel fiber (CFB) is, for example, polyglutamic acid, polyaspartic acid or the like.
  • a preferred embodiment of the method for producing a multilayer polymer-coated crosslinked alginate gel fiber can be arbitrarily formed.
  • the third aspect is as follows. Performing a cross-linking reaction using a cell capable of producing an antibody, a physiologically active substance, etc., a chemically modified alginic acid derivative represented by the formula (I) and a chemically modified alginic acid derivative represented by the formula (II) according to the above embodiment [1].
  • This is a method for producing an antibody, a physiologically active substance, or the like using a polymer-coated cross-linked alginic acid gel fiber formed by coating a core layer containing the cross-linked alginic acid gel obtained in the above method with a cationic polymer.
  • the polymer-coated crosslinked alginate gel fiber is placed in a culture vessel, a medium is added to impregnate the polymer-coated crosslinked alginate gel fiber, and the culture is carried out to obtain an antibody, a physiologically active substance, or the like. It is a manufacturing method.
  • the polymer-coated crosslinked alginate gel fiber according to the above aspect [3] is the polymer-coated crosslinked alginate gel fiber according to any one of the above aspects ([1] to [1-17-5]).
  • the culture vessel is a vessel selected from the group consisting of, for example, a tissue culture plate, a triangular flask, a T-flask, a spinner flask, a culture bag, an animal cell culture tank, and the like. Yes; preferably a triangular flask or an animal cell culture tank.
  • any method such as static culture or shaking culture may be selected, and any method such as batch culture (batch culture), feed culture (fed batch culture), or continuous culture may be used. It is good, but feeding culture or continuous culture is preferable.
  • the temperature during culturing is, for example, in the range of about 28 ° C to about 39 ° C, for example, about 30 ° C to about. It is in the range of 37 ° C.
  • the stirring speed during culturing is, for example, about 50 to about 250 rpm, for example, about 125 rpm.
  • the culture conditions are, for example, a culture temperature in the range of about 28 ° C to about 39 ° C and a 5% CO 2 atmosphere. Incubate in a culture device at a stirring speed of about 125 rpm.
  • the culture period is, for example, 7 days, 14 days, 28 days, or 42 days. Or 56 days, or 70 days.
  • the culture temperature when described as "about", it may include values up to ⁇ 10% of the value, and in some embodiments up to ⁇ 20% of the value.
  • the cells capable of producing an antibody, a physiologically active substance, etc. contained in the core layer of the polymer-coated crosslinked alginate gel fiber are the cells described in the above aspects [1-3] to [1-3-5]. ], Which is the same as the cell capable of producing the antibody, physiologically active substance, etc. according to any one of the above items.
  • the cells capable of producing an antibody, a physiologically active substance, etc. contained in the core layer of the polymer-coated crosslinked alginate gel fiber are the cells described in the above aspects [1B-3] to [1B-3]. -19] The same as the cells capable of producing the antibody, physiologically active substance, etc. according to any one of the above items.
  • [3-7] A method for producing an antibody, a physiologically active substance, or the like, which comprises adding a cell proliferation inhibitor in any one of the above embodiments [3] to [3-5].
  • the third aspect is as follows. Performing a cross-linking reaction using a cell capable of producing an antibody, a physiologically active substance, etc., a chemically modified alginic acid derivative represented by the formula (I) and a chemically modified alginic acid derivative represented by the formula (II) according to the above embodiment [1].
  • This is a method for producing an antibody, a physiologically active substance, or the like using a multilayer polymer-coated crosslinked alginate gel fiber formed by sequentially coating a core layer containing the crosslinked alginate gel obtained in the above step with a cationic polymer and an anionic polymer.
  • an antibody and a physiologically active substance are obtained by placing the multilayer polymer-coated crosslinked alginate gel fiber in a culture vessel, adding a medium, impregnating the multilayer polymer-coated crosslinked alginate gel fiber, and culturing.
  • Etc. is a manufacturing method.
  • the multilayer polymer-coated crosslinked alginate gel fiber according to the embodiment [3B] is the multilayer polymer-coated crosslinked alginate gel fiber according to any one of the above embodiments ([1B] to [1B-18-6]).
  • the temperature at the time of culturing is, for example, in the range of about 28 ° C to about 39 ° C, for example, about 30 ° C to about. It is in the range of 37 ° C.
  • the stirring speed during culturing is, for example, about 50 to about 250 rpm, for example, about 125 rpm.
  • the culture conditions are, for example, a culture temperature in the range of about 28 ° C to about 39 ° C and a 5% CO 2 atmosphere. Incubate in a culture device at a stirring rate of about 125 rpm.
  • the culture period is, for example, 7 days, 14 days, 28 days, or 42 days. Or 56 days, or 70 days.
  • the cells capable of producing the antibody, bioactive substance, etc. contained in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber are the cells of the embodiments [1-3] to [1-3-]. 5], the same as the cell capable of producing the antibody, bioactive substance, etc. according to any one of [1B-3] to [1B-3-19].
  • [3B-7] A method for producing an antibody, a physiologically active substance, or the like, which comprises adding a cell proliferation inhibitor in any one of the above embodiments [3B] to [3B-5].
  • a preferred embodiment of the method for producing an antibody, a physiologically active substance, etc. can be arbitrarily formed. ..
  • the fourth aspect is the cationicity produced in the core layer of the polymer-coated crosslinked alginate gel fiber obtained by the method for producing an antibody according to any one of the above aspects [3] to [3-7].
  • the antibody that permeates the polymer layer is an antibody having an isotype selected from the group consisting of, for example, IgG, IgA, IgM, IgD, IgE, and the like.
  • a fourth B aspect is a cationic polymer layer and an anionic polymer produced in the core layer of a multilayer polymer-coated crosslinked alginate gel fiber in the method for producing an antibody according to the above-mentioned aspects [3B] to [3B-7].
  • the antibody that permeates the layer is an antibody having an isotype selected from the group consisting of, for example, IgG, IgA, IgM, IgD, IgE, and the like.
  • the fifth aspect is the cationicity produced in the core layer of the polymer-coated crosslinked alginate gel fiber obtained by the method for producing an antibody according to any one of the above aspects [3] to [3-7].
  • the molecular weight of the antibody permeating the polymer layer is, for example, about 45,000 to about 1,000,000 Da, about 3,000 to about 1,000,000 Da, about 20,000 to about 1,000,000 Da, about 20.
  • a fifth B aspect is produced in the core layer of a multilayer polymer coated crosslinked alginate gel fiber in the antibody production method according to the above aspects [3B] to [3B-7], and is a cationic polymer layer and anionic.
  • the molecular weight of the antibody that permeates the polymer layer is, for example, about 3,000 to about 1,000,000 Da, about 20,000 to about 1,000,000 Da, about 45,000 to about 1,000,000 Da, and about 20.
  • the molecular weight of insulin produced using MIN6 cells by the production method according to any one of the above aspects [3] to [3-7] is, for example, about 5. Insulin in the range of 000 to 10,000.
  • the sixth aspect of the sixth B is the physiology produced by using the bioactive substance-producing cells by the production method according to the above-mentioned aspects [3] to [3-7] and [3B] to [3B-7].
  • the molecular weight of the active substance is, for example, about 3,000 to about 1,000,000 Da, about 20,000 to about 1,000,000 Da, about 45,000 to about 1,000,000 Da, about 20,000. It is a bioactive substance in the range of about 400,000 Da, about 45,000 to about 400,000 Da, about 20,000 to about 200,000 Da, and about 45,000 to about 200,000 Da.
  • the antibody obtained in the method for producing an antibody according to any one of the above aspects [3] to [3-7] is, for example, muromonab using muromonab-CD3-producing CHO cells.
  • Arilokumab ixekizumab-producing CHO cells, ixekizumab, brodalmab-producing CHO cells, brodalmab, idalcizumab-producing CHO cells, idalcizumab, erotuzumab-producing CHO cells, erotuzumab, pembu Pembrolizumab using lorizumab-producing CHO cells, salilumab using salilumab-producing CHO cells, bezlotoxmab using bezlotoxmab-producing CHO cells, berimumab using berimmab-producing CHO cells, and dalatumumab using daratumumab-producing CHO cells.
  • the antibodies obtained in the antibody production method according to the above aspects [3] to [3-7] and [3B] to [3B-7] are muromonab-CD3, trussumab, and rituximab.
  • Antibodies such as enholtumab, sashitsuzumab, verantamab, roncastuximab, chisotumab, datpotamab, patritumab; antibodies with modified sugar chains such as mogamurizumab, benlarizumab, obinutsuzumab, inebirizumab, inebirizumab, It is a small molecule antibody composed of an antibody fragment such as.
  • the antibody obtained in the method for producing the antibody according to the above aspects [3] to [3-7] and [3B] to [3B-7] is a muromonab-CD3 producing CHO cell.
  • the antibodies obtained in the method for producing the antibodies according to the above embodiments [3] to [3-7] and [3B] to [3B-7] are muromonab-CD3 producing CHO cells and trussumab producing CHO cells.
  • Lithuximab-producing CHO cells Lithuximab-producing CHO cells, paribismab-producing CHO cells, infliximab-producing CHO cells, basiliximab-producing CHO cells, tosirizumab-producing CHO cells, gemtuzumab-producing CHO cells, bebashizumab-producing CHO cells, ibritsumomab-producing CHO cells, adalimumab-producing CHO cells, adalimumab-producing CHO cells , Ranibizumab-producing CHO cells, omalizumab-producing CHO cells, eculizumab-producing CHO cells, panitumumab-producing CHO cells, ustequinumab-producing CHO cells, golimumab-producing CHO cells, canaquinumab-producing CHO cells, denosumab-producing CHO cells, mogamurizumab-producing CHO cells, mogamurizumab-
  • CHO cells dinutuximab-producing CHO cells, fremanezumab-producing CHO cells, elenumab-producing CHO cells, kasiribimab-producing CHO Cells, imdevimab-producing CHO cells, aniflorumab-producing CHO cells, sotrobimab-producing CHO cells, ocrylizumab-producing CHO cells, naxistamab-producing CHO cells, aducanumab-producing CHO cells, tafacitamab-producing CHO cells, marjetuximab-producing CHO cells, poratsumab-producing CHO cells, poratsumab-producing CHO cells Producing CHO cells, Sashitsuzumab-producing CHO cells, Verantamab-producing CHO cells, Roncastsumib-producing CHO cells, Chisotsumab-producing CHO cells, Inevirizumab-producing CHO cells, Brinatsumomab-producing CHO cells,
  • the antibodies obtained in the method for producing an antibody according to the above-described embodiments [3] to [3-7] and [3B] to [3B-7] are: trussumab-producing CHO cells, rituximab-producing CHO cells, and infliximab.
  • Antibodies produced from CHO cells such as producing CHO cells, tosirizumab-producing CHO cells, adalimmab-producing CHO cells, nivormab-producing CHO cells, and anti-GPVI antibody-producing CHO cells; or tosirizumab-producing CHO cells or anti-GPVI antibody-producing CHO cells. It is an antibody produced from; or an antibody produced from tosirizumab-producing CHO cells.
  • alginic acid as a raw material for synthesizing chemically modified alginic acid derivatives represented by the formulas (I) and (II), alginic acid as a raw material for an alginic acid solution or an alginic acid gel that can be contained in the core layer, and anionic.
  • the alginic acid used to form the polymer layer will be described below.
  • the term alginic acid means at least one alginic acid (sometimes referred to as "alginic acids") selected from the group consisting of alginic acid, alginic acid esters, and salts thereof (for example, sodium alginate). do.
  • the alginic acid used may be of natural origin or synthetic, but is preferably of natural origin.
  • Preferred alginic acids are bioabsorbable polysaccharides extracted from brown algae such as Lessonia, Macrocystis, Laminaria, Ascophyllum, Derbilia, Ecklonia cava, Arame, Combu and D-mannuronic acid (M).
  • It is a polymer in which two kinds of uronic acids, L-gluuronic acid (G) and L-gluuronic acid (G), are linearly polymerized. More specifically, the homopolymer fraction of D-mannuronic acid (MM fraction), the homopolymer fraction of L-gluuronic acid (GG fraction), and the random arrangement of D-mannuronic acid and L-gluuronic acid.
  • MM fraction homopolymer fraction of D-mannuronic acid
  • GG fraction homopolymer fraction of L-gluuronic acid
  • It is a block copolymer in which the resulting fractions (M / G fractions
  • Alginic acid is a kind of natural polysaccharide produced by extracting from seaweed of brown algae and purifying it, and is a polymer obtained by polymerizing D-mannuronic acid (M) and L-gluuronic acid (G).
  • the composition ratio (M / G ratio) of alginic acid D-mannuronic acid and L-gluuronic acid, that is, the gel strength, differs mainly depending on the type of organism from which seaweeds are derived, and also depends on the place of growth and season of the organism. Affected, it ranges from a high G type with an M / G ratio of about 0.2 to a high M type with an M / G ratio of about 5.
  • the physicochemical properties of alginic acid may differ depending on the M / G ratio of alginic acid, the arrangement of M and G, and the like, and the preferred uses may differ. It is known that the gelling ability of alginic acids and the properties of the produced gel are affected by the M / G ratio, and generally, the gel strength increases when the G ratio is high. The M / G ratio also affects the hardness, brittleness, water absorption, flexibility, etc. of the gel. Therefore, the alginic acid used in the present invention should have an appropriate M / G ratio and an appropriate viscosity depending on the final intended use.
  • the industrial production method of alginic acid includes an acid method and a calcium method, but in the present invention, any method produced by any of the production methods can be used.
  • the quantitative value by the HPLC method is preferably contained in the range of 80 to 120% by mass, more preferably contained in the range of 90 to 110% by mass, and contained in the range of 95 to 105% by mass. More preferred.
  • those whose quantitative values by the HPLC method are included in the above range are referred to as high-purity alginic acid.
  • the alginic acid or a salt thereof used in the present invention is preferably high-purity alginic acid.
  • a commercial product for example, as the Kimika algin series, a product sold by Kimika Co., Ltd., preferably a high-purity food / pharmaceutical grade product can be purchased and used. Commercially available products can be further purified and used as appropriate. For example, low endotoxin treatment is preferred. As the purification method or the low endotoxin treatment method, for example, the method described in JP-A-2007-7425 can be adopted. In the present specification, the term "mass%" means w / w% or w / v%.
  • the salt of alginic acid in "alginic acid” used in the present invention is "monovalent metal salt of alginic acid", and hydrogen ions of D-mannuronic acid of alginic acid or carboxylic acid of L-gluuronic acid can be used as Na + or K +. It is a salt produced by ion exchange with monovalent metal ions such as. Specific examples of the monovalent metal salt of alginic acid include sodium alginate and potassium alginate, but sodium alginate is particularly preferable.
  • alginic acid may be referred to as (ALG) -COOH, with alginic acid as (ALG) and one of any carboxyl groups of alginic acid as -COOH.
  • the alginic acid used in the present invention has an appropriate weight average molecular weight according to its final intended use.
  • the weight average molecular weight (GPC) of alginic acid used in the present invention is, for example, 10,000 to 10 million; preferably 100,000 to 5 million; more preferably 150,000 to 3 million.
  • the alginate is sodium alginate.
  • the sodium alginate commercially available sodium alginate can be used.
  • the sodium alginate used in the examples described later is the sodium alginate of A-1, A-2, A-3, B-1, B-2, and B-3 described in the table below (sold by Mochida). Selected from (Pharmaceutical Co., Ltd.). The viscosity, weight average molecular weight and M / G ratio of each 1 w / w% aqueous solution of sodium alginate are shown in the table below.
  • the physical property values of the sodium alginate A-1, A-2, A-3, B-1, B-2, and B-3 were measured by the following various methods.
  • the measuring method is not limited to the method, but each physical property value may differ from the above depending on the measuring method.
  • Pretreatment method An eluent was added to the sample to dissolve it, and then filtered through a 0.45 ⁇ m membrane filter to obtain a measurement solution.
  • Da (Dalton) may be added as a unit in the molecular weights of alginic acid, alginic acid derivatives, crosslinked alginic acid, and crosslinked alginic acid.
  • the composition ratio (M / G ratio) of D-mannuronic acid and L-gluuronic acid of alginic acids differs mainly depending on the type of organism from which seaweeds are derived, and is also affected by the habitat and season of the organism. , From a high G type with an M / G ratio of about 0.2 to a high M type with an M / G ratio of about 5. It is known that the gelling ability of alginic acids and the properties of the produced gel are affected by the M / G ratio, and generally, the gel strength increases when the G ratio is high. The M / G ratio also affects the hardness, brittleness, water absorption, flexibility, etc. of the gel.
  • the M / G ratio of the alginic acids and / or salts thereof used is usually 0.1 to 4.0, in some embodiments 0.1 to 3.0, and in some embodiments 0.1 to 2. It is 0.0, 0.5 to 1.8 in some embodiments, and 0.8 to 1.2 in some embodiments. In another aspect, it is 0.1 to 0.5.
  • alginic acid used in the present invention should have an appropriate viscosity and an appropriate M / G ratio according to the final intended use.
  • the numerical range indicated by using “-” indicates a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively.
  • alginic acid ester and “alginate” used in the present specification are not particularly limited, but in order to react with a cross-linking agent, it is necessary that they do not have a functional group that inhibits the cross-linking reaction.
  • Preferred examples of the alginic acid ester include propylene glycol alginate.
  • Alginic acid can be in the form of, for example, a monovalent salt of alginic acid or a divalent salt of alginic acid.
  • the monovalent salt of alginic acid include sodium alginate, potassium alginate, ammonium alginate, and the like, preferably sodium alginate or potassium alginate, and more preferably sodium alginate.
  • the divalent salt of alginic acid include calcium alginate, magnesium alginate, barium alginate, strontium alginate, and the like.
  • Alginic acid is a high molecular weight polysaccharide and it is difficult to accurately determine its molecular weight, but it generally has a weight average molecular weight of 10 to 10 million, preferably 10,000 to 8 million, and more preferably 20,000 to. It is 3 million. It is known that in the measurement of the molecular weight of a polymer substance derived from a natural product, the value may differ depending on the measurement method.
  • the molecular weight of the alginic acid derivative of the present invention or alginic acid or a salt thereof is specified, it is the weight average molecular weight calculated by size exclusion chromatography (SEC) unless otherwise specified.
  • SEC size exclusion chromatography
  • the alginic acid or a salt thereof used in the present invention it is desirable to use one having an appropriate molecular weight distribution according to the final intended use.
  • the temperature is preferably 100,000 to 5 million. Yes, more preferably 150,000 to 3 million. In some embodiments, it is 500,000 to 3 million, more preferably 1 million to 2.5 million, and even more preferably 1 million to 2 million.
  • the absolute weight average molecular weight can be measured.
  • the weight average molecular weight (absolute molecular weight) measured by the GPC-MALS method is preferably 10,000 or more, more preferably 50,000 or more, still more preferably 60,000 or more, and preferably 1 million or less, more preferably 80. It is 10,000 or less, more preferably 700,000 or less, and particularly preferably 500,000 or less. The preferred range is 10,000 to 1,000,000, more preferably 50,000 to 800,000, and even more preferably 60,000 to 500,000.
  • a measurement error of about 10% to about 30% can occur. For example, if it is 500,000, the value may fluctuate in the range of 350,000 to 650,000, and if it is 1 million, the value may fluctuate in the range of 700,000 to 1.3 million. In the present specification, when "about” is described in the description of molecular weight measurement, a value up to ⁇ 10% of the value, and in some embodiments up to ⁇ 20% of the value may be included.
  • a polymer substance derived from a natural product does not have a single molecular weight, but is an aggregate of molecules having various molecular weights, so that it is measured as a molecular weight distribution having a certain width.
  • a typical measurement method is gel filtration chromatography.
  • Typical information on the molecular weight distribution obtained by gel filtration chromatography includes weight average molecular weight (Mw), number average molecular weight (Mn), and dispersion ratio (Mw / Mn).
  • the weight average molecular weight emphasizes the contribution to the average molecular weight of a polymer having a large molecular weight, and is expressed by the following formula.
  • the number average molecular weight is calculated by dividing the total weight of the macromolecules by the total number of macromolecules.
  • W is the total weight of the polymer
  • Wi is the weight of the i-th polymer
  • Mi is the molecular weight at the i-th elution time
  • Ni is the number of molecular weight Mi
  • Hi is the height at the i-th elution time. ..
  • the weight average molecular weight is a value calculated by a calibration curve using pullulan as a standard material after measuring the molecular weight by, for example, size exclusion chromatography (SEC) by a conventional method as shown in the above literature. can do.
  • the weight average molecular weight can be an absolute molecular weight measured by, for example, size exclusion chromatography (SEC) -MALS by a conventional method as shown in the above-mentioned literature.
  • the molecular weight of alginic acids can be measured according to a conventional method.
  • the molecular weight of alginic acid or a salt thereof in the present specification is a weight average molecular weight calculated by gel filtration chromatography unless otherwise specified.
  • a typical condition when gel filtration chromatography is used for molecular weight measurement for example, the condition of this example described later can be adopted.
  • the column for example, Superose6 Increase10 / 300 GL column (GE Healthcare Science Co., Ltd.) can be used, and as a developing solvent, for example, a 10 mmol / L phosphate buffer solution (pH 7.4) containing 0.15 mol / L NaCl.
  • bluedextran, tyroglobulin, ferritin, aldolase, conalbumin, obalbumin, ribonuclease A and aprotinin can be used as molecular weight standards.
  • the viscosity of alginic acid used in the present specification is not particularly limited, but when the viscosity is measured as an aqueous solution of 1 w / w% alginic acid, it is preferably 10 mPa ⁇ s to 1000 mPa ⁇ s, more preferably 50 mPa ⁇ s. It is s to 800 mPa ⁇ s.
  • the viscosity of the aqueous solution of alginic acid can be measured according to a conventional method.
  • a co-axis double-cylindrical rotational viscometer, a single cylindrical rotational viscometer (Brookfield type viscometer), a cone-plate type rotational viscometer (cone plate type viscometer), etc. of the rotational viscometer method are used. Can be measured. It is preferable to follow the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). More preferably, a cone plate type viscometer is used.
  • Alginic acids initially have a large molecular weight and high viscosity when extracted from brown algae, but in the process of drying and refining by heat, the molecular weight becomes small and the viscosity becomes low.
  • Alginic acids having different molecular weights can be produced by methods such as controlling conditions such as temperature in the production process, selecting brown algae as raw materials, and fractionating the molecular weight in the production process. Further, it is also possible to obtain alginic acid having a desired molecular weight by mixing with another lot of alginic acid having a different molecular weight or viscosity.
  • the alginic acid used herein is alginic acid that has not been treated with low endotoxin in some embodiments, or alginic acid that has been treated with low endotoxin in some other embodiments.
  • Low endotoxin means that the endotoxin level is low enough not to cause inflammation or fever. More preferably, it is alginic acid treated with low endotoxin.
  • the low endotoxin treatment can be performed by a known method or a method similar thereto.
  • the method of Suga et al. For purifying sodium hyaluronate see, for example, JP-A-9-324001
  • the method of Yoshida et al. For purifying ⁇ 1,3-glucan (eg, JP-A-8-269102).
  • William et al.'S method for purifying biopolymer salts such as alginate and gellan gum (see, eg, JP-A-2002-530440)
  • James et al.'S method for purifying polysaccharides eg, international publication.
  • Low endotoxin treatment is not limited to these, but for cleaning, filtration with filters (endotoxin removal filter, charged filter, etc.), ultrafiltration, columns (endotoxin adsorption affinity column, gel filtration column, column with ion exchange resin, etc.).
  • the endotoxin level can be confirmed by a known method, and can be measured by, for example, a method using Limulus reagent (LAL), a method using Endospecy (registered trademark) ES-24S set (Seikagaku Corporation), or the like. ..
  • LAL Limulus reagent
  • Endospecy registered trademark
  • ES-24S set Seikagaku Corporation
  • the endotoxin content of the alginic acids should be 500 endotoxin units (EU) / g or less when the endotoxin is measured by the Limulus reagent (LAL). Is more preferable, and more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and particularly preferably 30 EU / g or less.
  • EU endotoxin units
  • substantially free of endotoxin means that the endotoxin value measured by the Japanese Pharmacopoeia endotoxin test is within the above numerical range.
  • Sodium alginate treated with low endotoxin can be obtained from commercial products such as Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.) and PRONOVA TM UP LVG (FMCBioPolymer).
  • sodium alginate as a raw material for synthesizing the chemically modified alginic acid derivatives represented by the formulas (I) and (II) in the present specification, and an alginic acid solution or alginic acid that can be contained in the core layer.
  • the sodium alginate used as a raw material for the gel is not particularly limited, but is, for example, sodium alginate A-1, A-2, A-3, B-1, B-2, or B shown in Table 8 above. It is possible to select from -3.
  • the sodium alginate used as a raw material for the alginate solution that can be used for the anionic polymer layer is not particularly limited, and is, for example, the sodium alginate A-1 shown in Table 8 above. It is possible to select from A-2, A-3, B-1, B-2, or B-3.
  • the concentration of the alginic acid solution (also referred to as sodium alginate solution) prepared using the sodium alginate is, for example, in the range of about 0.1 to about 3.3% by weight.
  • the sodium alginate used as a raw material for synthesizing the chemically modified alginic acid derivatives represented by the formulas (I) and (II) is preferably A-2, A-3, B-2 shown in Table 8 above. Or B-3, more preferably A-2 or A-3.
  • the concentration of the sodium alginate solution used in the synthesis of the chemically modified alginic acid derivative represented by the formulas (I) and (II) is preferably in the range of 1.5 to 2.0% by weight.
  • the alginate solution that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber, or the sodium alginate used for preparing the alginic acid solution used for forming the alginate gel is preferable.
  • the concentration of the alginic acid solution prepared using the sodium alginate is preferably in the range of about 0.3 to about 1.5% by weight.
  • the sodium alginate used for preparing an alginic acid solution that can be used for forming an anionic polymer layer of a multilayer polymer-coated crosslinked alginate gel fiber is preferably A-2 or B- shown in Table 8 above. It is 2.
  • the concentration of the alginic acid solution prepared using the sodium alginate is, for example, about 0.01 to about 5.0% by weight, about 0.05 to about 1.0% by weight, and about 0.1 to about 0. The concentration is in the range of 5% by weight, about 0.15 to about 0.4% by weight, and the like.
  • the alginic acid solution means a solution in which alginic acid is dissolved in a solvent.
  • the solvent is not particularly limited, and examples thereof include a medium, a cell culture medium, a culture solution, an isotonic buffer solution, water, a phosphate buffered saline (PBS), and a physiological saline solution.
  • PBS phosphate buffered saline
  • sodium alginate solution When sodium alginate is dissolved in a solvent, it is called a sodium alginate solution.
  • the solution of the chemically modified alginic acid derivatives represented by the formulas (I) and (II) used for forming the core layer of the polymer-coated cross-linked alginic acid gel fiber and the multilayer polymer-coated cross-linked alginic acid gel fiber, the alginic acid solution is also possible to mix a collagen solution, a medium, a culture solution and the like.
  • the solution of the chemically modified alginic acid derivative represented by the formula (I) or the formula (II) and the solvent used for preparing the alginic acid solution are as described below.
  • the chemically modified alginate derivatives herein are reactive in the Huisgen reaction described below via an amide bond and a divalent linker to any one or more carboxyl groups of the alginic acid.
  • a group or a reactive group complementary to the reactive group has been introduced. More specifically, the following formula (I): [Definition of (ALG), Akn-L 1- , and -NH-CO- in formula (I) is the same as the definition in the first aspect described above], and the alginic acid derivative represented by the following. Equation (II): It is an alginic acid derivative represented by [the definitions of (ALG), -L2-, and -NH-CO- in the formula ( II ) are the same as the definitions in the first aspect described above].
  • the divalent linker (-L 1- or -L 2- ) can be specifically selected and used from the divalent linkers described in the above embodiments.
  • the hydrogen atom of the imino group (-NH-) is replaced with a methyl group -N (Me) -CO-. It is possible to base it.
  • the bonding mode between the linker (-L 1- , -L 2- ) and alginic acid is -NH-CO- bonding or -N (Me). ) -CO-bonds; preferably -NH-CO- bonds.
  • the -NH-CO- bond or the -CO- of the -N (Me) -CO- bond is derived from the carboxyl group of alginic acid.
  • the chemically modified alginic acid derivative represented by the formula (I) or the formula (II) can be produced, for example, by the method for synthesizing the chemically modified alginic acid derivative described later.
  • the weight average molecular weight of the chemically modified alginic acid derivative represented by the formula (I) of the present specification measured by the gel filtration chromatography method is in the range of about 100,000 Da to about 3 million Da; preferably about 300,000 Da to. It is in the range of about 2.5 million Da, more preferably in the range of about 500,000 Da to about 2 million Da. Further, the weight average molecular weight of the chemically modified alginic acid derivative represented by the formula (II) measured by the gel filtration chromatography method is in the range of about 100,000 Da to about 3 million Da; preferably about 300,000 Da to about 250. It is in the range of 10,000 Da, more preferably in the range of about 500,000 Da to about 2 million Da.
  • the Akn-L 1 -NH-group of the formula (I) need not be bound to all the carboxyl groups of the alginic acid constituent unit, and the N 3 -L 2- of the formula (II).
  • the NH-group does not have to be attached to all the carboxyl groups of the alginic acid building block.
  • the N3-L2-NH-group of the formula (II) is a complementary reactive group.
  • the Akn - L1 - NH-group of the formula (I) is a complementary reactive group.
  • the introduction rate of the reactive group of the chemically modified alginic acid derivative represented by the formula (I) is, for example, in the range of about 0.1 to about 30 mol%; preferably about 0.3 to about 20 mol. %; More preferably, it is in the range of about 0.5 to about 10 mol%.
  • the introduction rate of the reactive group of the chemically modified alginic acid derivative represented by the formula (II) is, for example, in the range of about 0.1 to about 30 mol%; preferably in the range of about 0.3 to about 20 mol%. More preferably, it is in the range of about 0.5 to about 15 mol%.
  • the introduction rate of the reactive group or the complementary reactive group is expressed as a percentage of the number of uronic acid monosaccharide units into which each reactive group is introduced among the uronic acid monosaccharide units which are the repeating units of alginic acid. The value.
  • the introduction rate of each reactive group or complementary reactive group can be determined by the method described in Examples described later.
  • the cyclic alkyne group (Akn) in the formula (I) and the azide group in the formula (II) form a triazole ring by the Huisgen reaction, thereby forming a crosslink.
  • the chemically modified alginic acid derivative represented by the formula (I) or the formula (II) forms a monovalent salt (for example, sodium salt, etc.) at any carboxyl group in the molecule. Things are also included.
  • the chemically modified alginic acid derivatives represented by the formulas (I) and (II) are used for forming the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber and the multilayer polymer-coated crosslinked alginate gel fiber. It can also be used to form an anionic polymer layer in a multi-layer polymer coated crosslinked alginate gel fiber.
  • the Huisgen reaction (1,3-dipolar addition cyclization reaction) is a condensation reaction between compounds having a terminal azide group and a terminal alkyne group as shown in the following formula.
  • a disubstituted 1,2,3-triazole ring is obtained in good yield, and it has a feature that no extra by-product is generated. It is considered that a 1,4- or 1,5-disubstituted triazole ring can be formed in the reaction, but a regioselective triazole ring can be obtained by using a copper catalyst (Cu catalyst).
  • Cu catalyst copper catalyst
  • the Huisgen reaction is an azide compound having substituted primary azide, secondary azide, tertiary azide, aromatic azide, etc., and a terminal or cyclic alkyne which is a complementary reactive group of the azide group.
  • a compound having a group can be used.
  • various functional groups for example, ester group, carboxyl group, alkenyl group, hydroxyl group, amino group, etc. should be substituted in the reaction substrate. Is possible.
  • 1,2,3-triazoles are short-term, easily, and efficiently without the use of copper catalysts to produce unwanted by-products and avoid copper-catalyzed cytotoxicity.
  • the cyclic alkyne group (cyclooctyl group) according to the above embodiment [1] is used as the alkyne group of the Huisgen reaction.
  • the chemically modified alginic acid derivative represented by the formula (I) or the formula (II) is represented by the following reaction formula (AM-1) (Akn-L 1 ).
  • the amine represented by the formula (AM-2) N 3 -L 2 -NH 2 : -L 2 ).
  • -Is the same as the definition in the above aspect [1]) can be produced by a condensation reaction using an arbitrary carboxyl group of alginic acid and an arbitrary condensing agent.
  • the detailed conditions for each reaction are the same as those described in International Publication No. 2019/240219.
  • the introduction rate of the amine represented by the formula (AM-1) or the formula (AM-2) (the formula (in the above embodiment).
  • (I) or the introduction rate of the reactive group of the chemically modified alginic acid derivative represented by the formula (II) has the same meaning), by considering the properties of the amine and the like, the following (i) to (v) and the like can be obtained. Adjustment is possible by appropriately selecting and combining reaction conditions.
  • the following shows a more specific method for producing an amine used in the present specification among the amines represented by the formula (AM-1) or the formula (AM-2).
  • Z6, x7a, y7a, z7a, v7a, x7b, y7b, z7b, v7b, a1, b1, a2, b2, a3, b3, a4, b4, a5 and a6 are defined in the above embodiment [1].
  • RA C 1 to 6 alkyl groups such as methyl group, ethyl group, etc .
  • P 1 is -C (O) O-tertBu group, -C (O) O-Bn group,- It is a protective group for an amino group selected from 3 C (O) CH groups, 3 -C (O) CF groups, -SO 2 Ph, -SO 2 PhMe group, -SO 2 Ph (NO 2 ) group, and the like.
  • the protecting / deprotecting of the protecting group P1 is carried out by a method known in the literature, for example, "Protective Groups in Organic Synthesis 4th Edition", 4th edition. In 2007, protection / deprotection can be performed according to the deprotection method described in the textbook of John Wiley & Sons, Greene et al.
  • condensation reaction when described as a condensation reaction, it means a reaction similar to the above-mentioned condensation reaction.
  • a condensate is obtained by carrying out a condensation reaction using a compound of the formula (SM-A) and a compound of the formula (RG-A1). Subsequently, after addition of bromine, an alkyne group is formed by performing a debromination reaction using a base such as tert-BuOK. Subsequently, by deprotecting the protecting group P 1 , the amine represented by the formula (AM-1-1A) or a salt thereof can be produced.
  • Step 2> A condensation reaction is carried out using the compound of the formula (SM-A2) and the compound of the formula (RG-A2) obtained by the same method as in [Production Method A] ⁇ Step 1>, followed by protection. By deprotecting the group P 1 , the amine represented by the formula (AM-1-1B) or a salt thereof can be produced.
  • a condensate is obtained by carrying out a condensation reaction using the compound of the formula (SM-B) and the compound of the formula (RG-B1). Subsequently, by deprotecting the protecting group P1, the amine represented by the formula (AM- 1-2 ) or a salt thereof can be produced.
  • a condensation reaction is carried out using the compound of the formula (SM-D) and the compound of the formula (RG-D1) to obtain a condensed product. Subsequently, by deprotecting the protecting group P1, the amine represented by the formula (AM- 1 -D) or a salt thereof can be produced.
  • ⁇ Step 1> Using the compound of the formula (SM-F) and the compound of the formula (RG-F1), a condensation reaction is carried out to obtain a condensed product. Subsequently, by deprotecting the protecting group P1, the amine represented by the formula (AM- 1 -F) or a salt thereof can be produced.
  • ⁇ Step 2> Using the compound of the formula (SM-F) and the compound of the formula (RG-F2), a condensation reaction is carried out to obtain a condensed product. Subsequently, in the presence of a base such as sodium hydroxide, the ester group is hydrolyzed in a solvent such as methanol, ethanol, tetrahydrofuran, water, etc.
  • ⁇ Step 1> Using the compound of the formula (SM-G1) and the compound of the formula (RG-G1-1), a condensation reaction is carried out to obtain a condensed product. Subsequently, by deprotecting the protecting group P1, the amine represented by the formula (AM- 1 -G1) or a salt thereof can be produced.
  • ⁇ Step 2> Using the compound of the formula (SM-G1) and the compound of the formula (RG-G1-2), a condensation reaction is carried out to obtain a condensed product. Subsequently, the carboxylic acid represented by the formula (IM-G1) or a salt thereof can be produced by hydrolyzing the ester group.
  • ⁇ Step 3> [Production Method G] Using the compound of the compound of the formula (IM-G1) and the compound of the formula (RG-G1-3) obtained in ⁇ Step 2>, a condensation reaction is carried out to obtain a condensed product. .. Subsequently, by deprotecting the protecting group P1, the amine represented by the formula (AM- 1 -G1) or a salt thereof can be produced. ⁇ Step 4> Using the compound of the formula (SM-G2) and the compound of the formula (RG-G2-1), a condensation reaction is carried out to obtain a condensed product. Subsequently, by deprotecting the protecting group P1, the amine represented by the formula (AM- 1 -G2) or a salt thereof can be produced.
  • ⁇ Step 5> Using the compound of the formula (SM-G2) and the compound of the formula (RG-G2-2), a condensation reaction is carried out to obtain a condensate. Subsequently, the carboxylic acid represented by the formula (IM-G2) or a salt thereof (for example, a lithium salt, a sodium salt, a potassium salt, etc.) can be produced by hydrolyzing the ester group.
  • ⁇ Step 6> [Production Method G] Using the compound of the compound of the formula (IM-G2) and the compound of the formula (RG-G2-3) obtained in ⁇ Step 5>, a condensation reaction is carried out to obtain a condensed product. .. Subsequently, by deprotecting the protecting group P1, the amine represented by the formula (AM- 1 -G2) or a salt thereof can be produced.
  • ⁇ Step 1> Using the compound of the formula (SM-H) and the compound of the formula (RG-H1), a method known in the literature, for example, "European Journal of Organic Chemistry, 2014 (6), p1280-1286; 2014”. ], Etc., in the presence of (i) PPh 3 and N 2 (CO 2 CHMe 2 ) 2 reagents in a solvent that does not participate in the reaction, such as tetrahydrofuran, followed by a Mitsunobu reaction [ Production Method F] Similar to the method described in ⁇ Step 2>, a compound represented by the formula (IM-H1) or a salt thereof (for example, a lithium salt, a sodium salt, a potassium salt, etc.) is subjected to hydrolysis. ) Can be manufactured.
  • ⁇ Step 2> [Production Method H] A condensation reaction is carried out using the compound of the formula (IM-H1) and the compound of the formula (RG-H2) obtained in ⁇ Step 1> to obtain a condensed product. Subsequently, by deprotecting the protecting group P1, the amine represented by the formula (AM- 2 -H) or a salt thereof can be produced.
  • ⁇ Step 1A> [Production Method H] A condensation reaction is carried out using the compound of the formula (IM-H1) and the compound of the formula (RG-I1) obtained in ⁇ Step 1> to obtain a condensed product. Subsequently, by deprotecting the protecting group P1, the amine represented by the formula (AM-2- I ) or a salt thereof can be produced.
  • the formula (IM-J1) can be produced by carrying out a condensation reaction using the compound of the formula (SM-J) and the compound of the formula (RG-J1).
  • ⁇ Step 2> [Production Method J] Using the compound of the formula (IM-J1) obtained by ⁇ Step 1>, a method known in the literature, for example, "Organometrics, 29 (23), p6619-6622; 2010". Etc., according to the method described in the above, the formula (AM-2-J) is obtained by reacting NaN 3 in a solvent not involved in the reaction such as dimethyl sulfoxide to introduce an azide group, and then deprotecting the protecting group P1. ), Or a salt thereof.
  • ⁇ Step 1> The formula (IM-K) can be produced by carrying out a condensation reaction using the compound of the formula (SM-K) and the compound of the formula (RG-K).
  • ⁇ Step 2> [Manufacturing method K] Using the compound of the formula (IM-K) obtained by ⁇ Step 1>, the same reaction as in [Manufacturing method J] ⁇ Step 2 > and the deprotection of the protecting group P1 can be performed. By doing so, an amine represented by the formula (AM-2-K) or a salt thereof can be produced.
  • a condensation reaction is carried out using the compound of the formula (SM-L) and the compound of the formula (RG-L1) to obtain a condensed product. Subsequently, by deprotecting the protecting group P1, the amine represented by the formula (AM- 2 -L) or a salt thereof can be produced.
  • a condensation reaction is carried out using the compound of the formula (SM-L) and the compound of the formula (RG-M) to obtain a condensed product. Subsequently, by deprotecting the protecting group P1, the amine represented by the formula (AM- 2 -M) or a salt thereof can be produced.
  • C ( O)-, -CONH-, -O-, -NH-, -S-, cycloalkyl ring having 3 to 8 carbon atoms, benzene ring, heterocyclic ring (pyridine ring, piperidine ring, piperazine ring, etc.) 5-6 membered aromatic heterocycle or 5-6 membered non-aromatic heterocyclic ring), etc. may be replaced by a plurality of groups (for example, 1 to 10 or 1 to 5); the straight chain.
  • Halogen atom eg, fluorine atom, chlorine atom, bromine atom, iodine atom, etc.
  • a method known in the literature for example, "Experimental Chemistry Course 5th Edition, Each Book, 2007, Maruzen”, “Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 3rd” Edition (Edited by Richard C. Larock), 2018 ”,“ Strategic Applications of Named Reactions in Organic Synthesis, (Edited by Laszlo Kurti, Barbara Czako), Academic Press, 2005 ”, etc. Allows the desired amine to be produced.
  • the amine represented by the formula (AM-1) or the formula (AM-2) (including the lower formula of each formula) is a pharmaceutically acceptable salt (for example, an acid addition salt; For example, hydrochloride, hydrobromide, sulfate, acetate, trifluoroacetate, p-toluenesulfonate, etc.) may be formed.
  • a pharmaceutically acceptable salt for example, an acid addition salt; For example, hydrochloride, hydrobromide, sulfate, acetate, trifluoroacetate, p-toluenesulfonate, etc.
  • the compounds in the present specification can form salts, and according to a conventional method, for example, a solution containing an appropriate amount of acid or base is mixed to form a desired salt, and then the salt is separated and collected by filtration. It can be obtained by distilling off the mixed solvent.
  • a solution containing an appropriate amount of acid or base is mixed to form a desired salt, and then the salt is separated and collected by filtration. It can be obtained by distilling off the mixed solvent.
  • the amine represented by the formula (AM-1) or the formula (AM-2) (including the lower formulas of each formula) or a salt thereof is a solvate with a solvent such as water, ethanol, glycerol and the like.
  • a solvent such as water, ethanol, glycerol and the like.
  • variable substituent when a variable substituent is substituted on a cyclic group, it means that the variable substituent is not bonded to a specific carbon atom of the cyclic group.
  • the variable substituent Rs in the following formula A means that it can be substituted with any of the carbon atoms i, ii, iii, iv or v in the formula A.
  • cross-linked alginate gel In the present specification, the cross-linked alginate gel contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber is the chemically modified alginate described in the above-mentioned "2. Chemically modified alginate derivative".
  • cross-linked alginic acid gels with cross-linking also referred to as cross-linked alginic acid or chemically cross-linked alginic acid).
  • the crosslinked alginate gel contained in the core layer of each of the fibers is allowed to coexist with a chemical crosslink by a triazole ring formed by performing a Huisgen reaction (crosslinking reaction) as a crosslink, and a divalent metal ion (for example, calcium ion or the like). It is a crosslinked alginate gel containing both of the ionic crosslinks formed thereby.
  • the crosslinked alginate gel contained in the core layer of each of the fibers is a crosslinked alginate gel containing a chemical crosslink by a triazole ring formed by performing a Huisgen reaction (crosslinking reaction) as a crosslink.
  • the crosslinked alginate gel contained in the core layer of the polymer-coated crosslinked alginate gel fiber and the multilayer polymer-coated crosslinked alginate gel fiber is a chemically modified alginate derivative represented by the above formula (I) and the above formula (II). It is obtained by carrying out a Huisgen reaction (crosslinking reaction) in which a chemical crosslink is formed between the derivatives. Alternatively, it can be obtained by allowing a divalent metal ion to coexist with the chemically modified alginic acid derivative represented by the formula (I) and the formula (II) to form an ion crosslink between the derivatives.
  • the Huisgen reaction (crosslinking reaction) is carried out using the chemically modified alginic acid derivatives represented by the formulas (I) and (II) to form chemical crosslinks between the derivatives, and further divalent metal ions.
  • the cross-linking is formed means the chemically modified alginic acid derivative represented by the above formula (I) and the above-mentioned formula (II).
  • a chemical bridge chemical bond
  • the chemically modified alginic acid derivatives represented by the formulas (I) and (II) are coexisting the divalent metal ion with the chemically modified alginic acid derivative represented by the formula (II).
  • an ion bridge (ion bond) is formed between the derivatives, or both a chemical bridge by the Huisgen reaction and an ion bridge by a divalent metal ion are formed. It also means that an ionic crosslink is formed in alginic acid by coexisting divalent metal ions with alginic acid (sodium alginate or the like).
  • an ion crosslink is formed, and the time for forming an ion-crosslinked alginate gel is, for example, instantaneous (for example). 1 to 5 seconds) to several hours (for example, 1 to 3 hours). Further, the time for the Huisugen reaction to proceed between the chemically modified alginate derivatives represented by the formula (I) and the formula (II) to form a chemical crosslink and become a chemically crosslinked alginate gel is, for example, several seconds to 24. Hours, seconds to 12 hours, or seconds to 30 minutes.
  • the divalent metal ion used to obtain the crosslinked alginate gel is not particularly limited, and examples thereof include calcium ion, magnesium ion, barium ion, strontium ion, zinc ion and the like, and calcium ion and barium ion are preferable. Alternatively, it is a strontium ion, more preferably a calcium ion or a barium ion.
  • the solution containing divalent metal ions is not particularly limited, but for example, a solution containing calcium ions (for example, an aqueous solution such as a calcium chloride aqueous solution, a calcium carbonate aqueous solution, a calcium gluconate aqueous solution, etc.) and a solution containing barium ions (for example).
  • a solution containing calcium ions for example, an aqueous solution such as a calcium chloride aqueous solution, a calcium carbonate aqueous solution, a calcium gluconate aqueous solution, etc.
  • a solution containing barium ions for example.
  • An aqueous solution such as a barium chloride aqueous solution
  • a solution containing strontium ions for example, an aqueous solution such as a strontium chloride aqueous solution
  • the divalent metal ion concentration (for example, calcium ion or barium ion concentration) of the solution containing the divalent metal ion is not particularly limited, but is, for example, in the range of about 1 mM to about 1 M, or in the range of about 10 to about 500 mM. It is preferably about 10 to about 100 mM.
  • the solvent used when preparing a solution containing divalent metal ions is not particularly limited, but for example, tap water, pure water (for example, distilled water, ion-exchanged water, RO water, RO-EDI water, etc.), and the like.
  • Examples thereof include ultrapure water (MilliQ water), medium, cell culture medium, culture solution, phosphate buffered physiological saline (PBS), physiological saline and the like; preferably physiological saline or ultrapure water.
  • the reaction of ionic cross-linking is instantaneous and reversible, whereas the reaction of chemical cross-linking is a relatively mild condition. The reaction proceeds slowly and is irreversible.
  • the cross-linked alginate gel of the present invention can be efficiently produced. For example, using the apparatus XX in "11.
  • a mixed solution of chemically modified alginic acid derivatives represented by the formulas (I) and (II) is mixed with a divalent metal ion.
  • ionic cross-linking is formed, and a fiber-shaped cross-linked alginate gel can be instantly produced.
  • a Huisgen reaction crosslinking reaction
  • a (fiber) shaped crosslinked alginate gel can be obtained.
  • the physical characteristics of the crosslinked alginate gel are, for example, changing the concentration of the aqueous solution containing the divalent metal ion to be used (for example, calcium chloride aqueous solution) or the introduction rate of the reactive group introduced into the chemically modified alginate derivative. It can be adjusted by the method.
  • the chemically modified alginic acid derivatives represented by the formulas (I) and (II) are contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber of the present invention.
  • the crosslinked alginate gel can be produced in the form of a fiber (fiber) (also referred to as “crosslinked alginate gel fiber”).
  • fiber also referred to as “crosslinked alginate gel fiber”.
  • an alginic acid (for example, sodium alginate) solution is added to a mixed solution of chemically modified alginic acid derivatives represented by the formulas (I) and (II). Can be done.
  • chemical cross-linking (Huisugen reaction) is used as one of the methods for strengthening the fiber structure (for example, obtaining long-term stability, etc.).
  • a cross-linked alginate gel fiber having both ionic cross-linking and chemical cross-linking prepared as described above the divalent metal ions forming the ionic cross-linking gradually and reversibly. It becomes a cross-linked alginate gel fiber that is released and only the chemical cross-linking remains, but the gel structure is maintained by the irreversible chemical cross-linking, and stable and continuous culture is possible.
  • the crosslinked alginic acid gel formed by performing a crosslinking reaction using the chemically modified alginic acid derivatives represented by the formulas (I) and (II) of the present invention is not particularly limited, and is, for example, a collagen solution, a collagen gel, and the like.
  • Other components such as a medium, a medium for cell culture, a culture solution, methyl cellulose, a sucrose solution, an alginic acid solution, an alginate gel, and the like can be contained.
  • alginate gel means an alginate gel in which an ion crosslink is formed by coexisting divalent metal ions with alginic acid (for example, sodium alginate) or a solution thereof. ..
  • the cross-linked alginate gel can be obtained by mixing the chemically modified alginic acid derivatives represented by the formulas (I) and (II) and carrying out a Huisgen reaction. ..
  • the crosslinked alginate gel forms a three-dimensional network structure via chemical crosslinking (crosslinking by a triazole ring formed from an alkyne group and an azido group).
  • Preferred chemically modified alginate derivatives are those that improve the stability of the crosslinked alginate gel after crosslinking.
  • the physical characteristics of the crosslinked alginic acid gel can be adjusted, for example, by the introduction rate of each reactive group in the chemically modified alginic acid represented by the formula (I) or the formula (II) as a raw material.
  • the cross-linked alginate gel of some embodiments is described in the following formula (III-L): [In formula (III-L), -CONH- and -NHCO- at both ends represent amide bonds via any carboxyl group of alginic acid; -L 1- , -L 2- , and -X- represent. It is a cross-linked alginate gel cross-linked via a group represented by [the same as the definition in the above aspect [1-12]].
  • the mixing ratio of the chemically modified arginic acid derivative represented by the formula (I) to the chemically modified arginic acid derivative represented by the formula (II) in producing the crosslinked arginic acid gel is the formula (I).
  • the weight ratio of the chemically modified alginic acid derivative represented by the formula (II) to, for example, 1: 1.0 to 4.0 or 1: 1.0 to 3.0. , Or 1: 1.0 to 2.0, or 1: 1.0 to 1.5, or 1: 1; preferably 1: 1.0 to 3.0.
  • the mixing ratio of the chemically modified arginic acid derivative represented by the formula (II) to the chemically modified arginic acid derivative represented by the formula (I) in producing a crosslinked arginic acid gel is the formula (II).
  • the weight ratio of the chemically modified alginic acid derivative represented by the formula (I) to, for example, 1: 1.0 to 4.0 or 1: 1.0 to 3.0. , Or 1: 1.0 to 2.0, or 1: 1.0 to 1.5, or 1: 1.
  • the mixing ratio of the chemically modified arginic acid derivative represented by the formula (I) to the chemically modified arginic acid derivative represented by the formula (II) in preparing the crosslinked arginic acid gel is more preferable.
  • the ratio of the introduction rate (mol%) of the reactive group of the chemically modified alginic acid derivative represented by the formula (I) to the chemically modified alginic acid derivative represented by the formula (II) is, for example, 1: 1.0 to 4. 0, or 1: 1.0 to 3.0, or 1: 1.0 to 2.0, or 1: 1.0 to 1.5, or 1: 1; preferably 1: 1.0 to 1. It is 3.0.
  • the mixing ratio of the chemically modified arginic acid derivative represented by the formula (II) to the chemically modified arginic acid derivative represented by the formula (I) in preparing the crosslinked arginic acid gel is more preferable.
  • the ratio of the introduction rate (mol%) of the reactive group of the chemically modified alginic acid derivative represented by the formula (II) to the chemically modified alginic acid derivative represented by the formula (I) is, for example, 1: 1.0 to 4. It is 0, or 1: 1.0 to 3.0, or 1: 1.0 to 2.0, or 1: 1.0 to 1.5, or 1: 1.
  • the crosslinked alginic acid gel does not need to have all the carboxyl groups of the constituent units of alginic acid having the crosslink of the above formula (III-L).
  • the introduction rate (also referred to as the crosslinking rate) of the crosslinking represented by the above formula (III-L) is, for example, about 0.1 to about 80%, about 0.3 to about 60%, and about 0. It ranges from 5 to about 30%, or about 1.0 to about 10%.
  • the solution of the alginic acid derivative represented by the above formula (I) or the formula (II) in the Huisgen reaction for obtaining the crosslinked alginic acid gel contained in the core layer of the polymer-coated crosslinked alginic acid gel fiber and the multilayer polymer-coated crosslinked alginic acid gel fiber of the present invention.
  • the concentration of is, for example, in the range of about 0.01 to about 1.5% by weight; preferably in the range of about 0.05 to about 1.0% by weight; more preferably about 0.08 to about 0.08% by weight. It is in the range of about 0.75% by weight.
  • the concentration of the alginic acid solution is, for example, in the range of 0% to about 1.98% by weight. Yes; preferably in the range of 0% to about 1.8% by weight; more preferably in the range of 0% to about 1.7% by weight.
  • the reaction temperature of the Huisgen reaction (crosslinked alginate gel, temperature at the time of producing the crosslinked alginate gel fiber) is usually an outside temperature of about 4 to about 60 ° C., preferably an outside temperature of about 15 to about 37 ° C. ..
  • the polymer-coated cross-linked alginate gel fiber contains cells capable of producing antibodies, physiologically active substances, etc., and chemically modified alginic acid derivatives represented by the above formulas (I) and (II). It means a fibrous (fibrous) structure obtained by coating a core layer containing a crosslinked alginate gel obtained by carrying out a crosslinking reaction with a cationic polymer (cationic polymer layer). The method for producing the coated crosslinked alginate gel fiber will be described later).
  • the polymer-coated crosslinked alginate gel fiber is a fiber-like (fibrous) structure including a core layer and a cationic polymer layer arranged outside the core layer.
  • the core layer contains cells capable of producing antibodies, physiologically active substances and the like, and a crosslinked alginic acid gel in which crosslinks are formed using the chemically modified alginic acid derivatives represented by the formulas (I) and (II).
  • the cationic polymer layer is a cationic polymer.
  • the polymer-coated crosslinked alginate gel fiber is a fiber-like (fibrous) structure including a core layer and a cationic polymer layer arranged outside the core layer.
  • the core layer contains cells capable of producing antibodies, physiologically active substances and the like, and a crosslinked alginate gel, and the crosslinked alginate gel uses chemically modified alginic acid derivatives represented by the formulas (I) and (II).
  • the cationic polymer layer is a cationic polymer, which comprises cross-linking obtained by carrying out a cross-linking reaction.
  • FIG. 1 shows a cross-sectional view of an example of a polymer-coated crosslinked alginate gel fiber formed by coating a crosslinked alginate gel fiber with a cationic polymer.
  • This polymer-coated crosslinked alginate gel fiber has an outer diameter of c and includes a core layer 5 having a diameter a and a cationic polymer layer 4 having a thickness b, and the core layer 5 produces an antibody, a physiologically active substance, and the like. It contains a cross-linked alginate gel containing cells 6.
  • the cross-linked alginic acid gel of the core layer 5 is a cross-linked alginic acid gel formed by performing a cross-linking reaction using chemically modified alginic acid derivatives represented by the formulas (I) and (II).
  • the core layer of the polymer-coated crosslinked arginic acid gel fiber of some embodiments is formed by carrying out a crosslinking reaction using the chemically modified alginic acid derivatives represented by the formulas (I) and (II) described in the above embodiment [1].
  • the crosslinked alginic acid gel there is no particular limitation as long as it does not have cytotoxicity, but it is not particularly limited, but collagen solution, collagen gel, medium, cell culture medium, culture solution, methyl cellulose, sucrose solution, alginic acid solution, alginate gel. , Etc.; preferably, a component selected from the group consisting of an alginic acid solution, an alginate gel, a medium, and a culture solution.
  • the "polymer-coated crosslinked alginic acid gel fiber” is a fibrous structure in which the outer diameter (c in FIG. 2) of the fiber is, for example, about 0.1 to about 2000 ⁇ m. Sometimes called "microfiber”.
  • the cross-sectional shape of the polymer-coated crosslinked alginate gel fiber in the direction perpendicular to the central axis is not limited to a circle, but may be an asymmetric structure or a deformed shape.
  • the cross-sectional shape may be circular, elliptical, or polygonal. It may have various shapes such as (for example, a triangle, a quadrangle, a pentagon, etc.), and is preferably a circular cross-sectional shape as shown in FIG.
  • the outer diameter of the polymer-coated crosslinked alginate gel fiber (in the case of non-circular shape, the major axis or the maximum diameter is regarded as the outer diameter) is, for example, about 0.1 to about 2000 ⁇ m, about 0.2 ⁇ m to about 2000 ⁇ m, and about 1 to about.
  • the range is 1000 ⁇ m, about 2 to about 500 ⁇ m, about 2 to about 200 ⁇ m, and the like.
  • the diameter of the core layer of the polymer-coated crosslinked alginate gel fiber is, for example, about 0.1 to about 2000 ⁇ m, about 0.2 ⁇ m to about 2000 ⁇ m, about 1 to about 1000 ⁇ m, about 2 to about 500 ⁇ m, about 2 to about 200 ⁇ m, and the like. It is a range.
  • the diameter of the cross section of the core layer is preferably less than the diameter of the fiber cross section and preferably 50% or more.
  • the thickness of the polymer layer is, for example, about 0.1 to about 200 ⁇ m, about 1 to about 200 ⁇ m, about 5 ⁇ m to about 200 ⁇ m, and the like.
  • the values of the diameter, outer diameter, and inner diameter of the core layer of the polymer-coated crosslinked alginate gel fiber are determined by, for example, preparing a fiber using a cationic polymer that develops fluorescent color in the polymer layer and using a retardation optical microscope. It is possible to measure from the image. It is expressed as the average value of the measured values at several points of the polymer-coated crosslinked alginate gel fiber.
  • the core layer and the polymer layer of the polymer-coated crosslinked alginate gel fiber usually have a substantially uniform thickness, and preferably each layer has a thickness uniformity within the range of ⁇ 10%.
  • the length of the polymer-coated crosslinked alginate gel fiber is not particularly limited, but is, for example, about 0.01 to about 100 m, or about 0.1 to about 75 m, or about 0.3 to about 50 m. ..
  • the core layer of the polymer-coated crosslinked alginate gel fiber of some embodiments contains a cell capable of producing an antibody, a physiologically active substance, or the like, and is chemically modified by the formulas (I) and (II). It can be formed using a mixed solution of alginic acid derivatives.
  • the concentration of the solution of the chemically modified alginic acid derivative represented by the formula (I) or the formula (II) is, for example, in the range of about 0.01 to about 1.5% by weight, respectively; preferably about 0. It ranges from 0.05 to about 1.0% by weight; more preferably from about 0.08 to about 0.75% by weight.
  • the concentration of the mixed solution of the chemically modified alginic acid derivatives represented by the formulas (I) and (II) is, for example, in the range of about 0.02 to about 2.0% by weight; preferably about 0.1. It is in the range of about 2.0% by weight; more preferably in the range of about 0.15 to about 1.5% by weight.
  • the concentration of the alginic acid is, for example, 0 to about 1.98% by weight. It is in the range; preferably in the range of 0 to about 1.8% by weight; more preferably in the range of 0 to about 1.7% by weight.
  • the chemically modified alginic acid derivatives represented by the formulas (I) and (II) containing cells capable of producing an antibody, a physiologically active substance, etc. used for forming the core layer of the polymer-coated crosslinked alginate gel fiber.
  • the chemically modified alginic acid derivatives represented by the formulas (I) and (II) containing cells capable of producing an antibody, a physiologically active substance, etc. used for forming the core layer of the polymer-coated crosslinked alginic acid gel fiber.
  • a mixed solution containing a cell capable of producing an antibody, a physiologically active substance, etc. used for forming a core layer of a polymer-coated crosslinked alginate gel fiber and a chemically modified alginic acid derivative represented by the formulas (I) and (II).
  • a mixed solution containing cells capable of producing an antibody, a physiologically active substance, etc. used for forming the core layer of a polymer-coated crosslinked arginic acid gel fiber and chemically modified arginic acid derivatives represented by the formulas (I) and (II).
  • the alginic acid solution is added to the mixture, the volume of the chemically modified alginic acid derivative represented by the formula (I) (v1) and the volume of the chemically modified alginic acid derivative represented by the formula (II) in the mixed solution to which the alginic acid solution is added.
  • chemically modified alginic acid derivatives represented by the formulas (I) and (II) containing cells capable of producing an antibody, a physiologically active substance, etc. used for forming the core layer of a polymer-coated crosslinked alginic acid gel fiber used for forming the core layer of a polymer-coated crosslinked alginic acid gel fiber.
  • the molecular weight of alginic acid (for example, sodium alginate, etc.) used for preparing the alginic acid solution is not particularly limited, but is measured by a gel filtration chromatography method (GPC method).
  • the weight average molecular weight obtained is, for example, in the range of about 150,000 Da to about 2,500,000 Da, in the range of about 300,000 Da to about 2,000,000 Da, in the range of about 700,000 Da to about 2,000,000 Da, and the like. Is.
  • chemically modified alginic acid derivatives represented by the formulas (I) and (II) containing cells capable of producing an antibody, a physiologically active substance, etc. used for forming the core layer of a polymer-coated crosslinked alginic acid gel fiber used for forming the core layer of a polymer-coated crosslinked alginic acid gel fiber.
  • the molecular weight of alginic acid (for example, sodium alginate, etc.) used for preparing the alginic acid solution is not particularly limited, but is measured by a gel filtration chromatography method (GPC method).
  • the weight average molecular weight is, for example, in the range of about 150,000 Da to about 2.5 million Da, in the range of about 300,000 Da to about 2.5 million Da, from about 700,000 Da to about 1.400,000 Da, about 800. It ranges from 000 Da to about 1,500,000 Da, about 1,400,000 to about 2,000,000 Da, about 1,500,000 to about 2,500,000, and the like.
  • the solvent used when preparing the solution of the chemically modified alginic acid derivative represented by the formula (I) or the formula (II), the alginic acid solution, etc. used when producing the core layer of the polymer-coated crosslinked alginic acid gel fiber is particularly limited.
  • examples thereof include media, cell culture media, culture media, isotonic buffers, phosphate buffered physiological saline (PBS), physiological saline and the like; preferably media, cell culture media, cultures and the like.
  • PBS phosphate buffered physiological saline
  • a solution, physiological saline or isotonic buffer phosphate buffered physiological saline or isotonic buffer.
  • Cationic polymer A polycation means a compound having two or more cationic groups in one molecule, and a cationic group means a cationic group or a group that can be induced by a cationic group.
  • a cationic group include an amino group; a monoalkylamino group such as a methylamino group and an ethylamino group; a dialkylamino group such as a dimethylamino group and a diethylamino group; an imino group; a guanidino group and the like.
  • the amino group may be a -NH 3+ group to which a proton is coordinated .
  • the cationic polymer means a polymer having two or more cationic groups in one molecule.
  • the cationic polymer include those obtained by polymerizing a monomer having a cationic group.
  • the cationic polymer preferably has a hydrophilicity that can be dissolved in water and has a property of being positively charged by dissociation of the cationic group in water.
  • a polymer having two or more amino groups in one molecule is particularly preferable.
  • the cationic polymer is formed by carrying out a cross-linking reaction using chemically modified alginic acid derivatives represented by the formulas (I) and (II), which include cells capable of producing an antibody, a physiologically active substance and the like.
  • the carboxyl group of the crosslinked alginate gel fiber and the cationic group of the cationic polymer interact electrostatically, and the surface of the crosslinked alginate gel fiber is coated with the cationic polymer.
  • the material is capable of increasing the strength of the crosslinked alginate gel fiber (see FIG. 2).
  • the cationic polymer the antibody, the physiologically active substance, etc.
  • the cationic polymer (polymer layer) covering the core layer It is preferably a substance that can be released to the outside of the polymer-coated crosslinked alginate gel fiber.
  • examples of the cationic polymer include polyamino acids (polymers of basic amino acids), basic polysaccharides (eg, chitosan, etc.), basic polymers (polymethylene-CO-guanidine (PMCG), polyallylamine).
  • PAA polyvinylamine
  • PVA polyethyleneimine
  • allylamine-diallylamine copolymer polyallylamine-maleic acid copolymer
  • cationic polymers are mentioned, and poly-L-ornithin (poly-L-ornithin) which is a polyamino acid is preferable.
  • PLO poly-D-ornithine
  • PDO poly-DL-ornithin
  • PDL poly-D-lysine
  • PLL poly-L-lysine
  • PLM poly-DL-lysine
  • PPA poly-L-arginine
  • Poly-D-Arginine (PDA), Poly-DL-Arginin, Poly-L-Homoarginine (PLHA), Poly-D-Homoarginine (PDHA), Poly-DL-Homoarginine, Poly-L-Histidine (PLH) ), Poly-D-histidine (PDH), and a cationic polymer selected from the group consisting of poly-DL-histidine; more preferably poly-L-ornithine, poly-L-lysine or polyallylamine; More preferably, it is poly-L-ornithine.
  • examples of the cationic polymer used when preparing a solution containing a cationic polymer include the polyamino acid, a basic polysaccharide, a basic polymer, and salts thereof (salt, hydrobromide). Etc.).
  • a commercially available product or a product prepared from a commercially available product can be used.
  • the degree of polymerization of the cationic polymer is not particularly limited, and examples thereof include a degree of polymerization of 50 to 6,000, a degree of polymerization of 50 to 2,000, and a degree of polymerization of 100 to 1,500. Be done.
  • the degree of polymerization is 130 to 1,300
  • the degree of polymerization is 50 to 1,800
  • chitosan for example, 60 to 6,000. Degree of polymerization.
  • the weight average molecular weight (Mw) of the cationic polymer is not particularly limited, but is, for example, in the range of 500 to 1,000,000, in the range of 1,000 to 500,000, and from 3,000 to. Within the range of 300,000, within the range of 5,000 to 100,000, within the range of 10,000 to 50,000, and the like.
  • the weight average molecular weight (Mw) of the cationic polymer can be measured by gel permeation chromatography (GPC).
  • poly-L-ornithin a commercially available poly-L-ornithine hydrobromide [for example, molecular weight: 70,000 to 150,000 (Fuji Film-manufactured by Wako Pure Chemical Industries, Ltd.), molecular weight: 15,000. ⁇ 30,000 to 30,000 to 70,000, 5,000 to 15,000 (manufactured by Sigma-Aldrich), etc.] can be used; for example, in the case of polyallylamine, a commercially available polyallylamine [eg, for example.
  • Chitosan one of the cationic polymers, is a deacetylase of chitin, and from the viewpoint of its water solubility, the degree of deacetylation thereof is, for example, in the range of 40 to 100% and in the range of 45 to 90%. , Or those in the range of 50 to 80% can be used.
  • the concentration of the solution containing the cationic polymer is not particularly limited, but may be any concentration as long as it can uniformly coat the surface of the alginate gel fiber, for example, about 0.01 to about 10.0% by weight, about 0. Concentrations of 0.01 to about 5.0% by weight and about 0.02 to about 1.0% by weight are mentioned, preferably from about 0.02 to about 5.0% by weight, more preferably from about 0.05 to about 0.05% by weight. The concentration is about 1.0% by weight.
  • the viscosity of the solution containing the cationic polymer is not particularly limited, but is, for example, in the range of 10.0 to 500.0 mPa ⁇ s, in the range of 20.0 to 300.0 mPa ⁇ s, and 50.0 to 200.0 mPa. ⁇ It is within the range of s.
  • the solvent of the solution containing the cationic polymer is not particularly limited as long as it can dissolve the cationic polymer, but for example, water (tap water, pure water (for example, distilled water, ion-exchanged water, RO water, RO). -EDI water, etc.), ultra-pure water (MilliQ water)), aqueous solutions of inorganic salts (phosphate buffered physiological saline (PBS), physiological saline, etc.), etc., and the amount of charge of the cationic polymer is increased. Ultra-pure water, water or physiological saline is preferable.
  • the multilayer polymer coated crosslinked alginate gel fiber is the cationic polymer layer of the polymer coated crosslinked alginate gel fiber described in "6.
  • Polymer coated crosslinked alginate gel fiber described above. It means a fibrous (fibrous) structure obtained by coating the outside of the above with an anionic polymer (anionic polymer layer). That is, a core containing a cell capable of producing an antibody, a physiologically active substance, or the like and a crosslinked alginic acid gel obtained by performing a crosslinking reaction using the chemically modified alginic acid derivatives represented by the formulas (I) and (II).
  • Polymer-coated crosslinked alginic acid obtained by coating the layer with a cationic polymer A fibrous structure obtained by coating the outside of the cationic polymer layer of the gel fiber with anionic kapolymer (multilayer polymer-coated crosslinked alginic acid). The method for manufacturing the gel fiber will be described later).
  • the multilayer polymer-coated crosslinked alginate gel fiber is fibrous (fibrous) including a core layer, a cationic polymer layer arranged outside the core layer, and an anionic polymer layer arranged outside the cationic polymer layer.
  • the core layer contains cells capable of producing antibodies, physiologically active substances and the like, and a crosslinked alginic acid gel in which crosslinks are formed using the chemically modified alginic acid derivatives represented by the formulas (I) and (II).
  • the cationic polymer layer is a cationic polymer
  • the anionic polymer layer is an anionic polymer.
  • the multilayer polymer-coated crosslinked alginate gel fiber is fibrous (fibrous) including a core layer, a cationic polymer layer arranged outside the core layer, and an anionic polymer layer arranged outside the cationic polymer layer.
  • the core layer contains cells capable of producing an antibody, a physiologically active substance, etc., and a crosslinked alginate gel, and the crosslinked alginate gel uses chemically modified alginate derivatives represented by the formulas (I) and (II).
  • the cationic polymer layer is a cationic polymer
  • the anionic polymer layer is an anionic polymer.
  • FIG. 8 shows a cross-sectional view of an example of a multilayer polymer coated crosslinked alginate gel fiber.
  • the multilayer polymer-coated crosslinked alginate gel fiber has an outer diameter of e and includes a core layer 5 having a diameter a, a cationic polymer layer 4 having a thickness b, and an anionic polymer layer 7 having a thickness d, and the core layer 5 has a core layer 5.
  • the cross-linked alginic acid gel of the core layer 5 is a cross-linked alginic acid gel formed by performing a cross-linking reaction using chemically modified alginic acid derivatives represented by the formulas (I) and (II).
  • the core layer of the multilayer polymer-coated crosslinked alginic acid gel fiber of some embodiments is subjected to a crosslinking reaction using the chemically modified alginic acid derivatives represented by the formulas (I) and (II) described in the above embodiment [1].
  • the formed crosslinked alginic acid gel there is no particular limitation as long as it does not have cytotoxicity, but it is not particularly limited, but collagen solution, collagen gel, medium, cell culture medium, culture solution, methyl cellulose, sucrose solution, alginic acid solution, alginic acid.
  • Other components such as gels can be included; preferably components selected from the group consisting of alginic acid solutions, alginate gels, media, culture solutions can be included.
  • the multilayer polymer-coated crosslinked alginate gel fiber is a fibrous structure in which the outer diameter (e in FIG. 8) of the fiber is, for example, about 0.1 to about 2000 ⁇ m. Sometimes called fiber.
  • the cross-sectional shape of the multilayer polymer-coated cross-linked alginate gel fiber in the direction perpendicular to the central axis is not limited to a circle, but may be an asymmetric structure or a deformed shape.
  • the cross-sectional shape may be circular, elliptical, or poly. It may have various shapes such as a polygon (for example, a triangle, a quadrangle, a pentagon, etc.), and is preferably a circular cross-sectional shape as shown in FIG.
  • the outer diameter of the multilayer polymer coated crosslinked alginate gel fiber (in the case of non-circular shape, the major axis or the maximum diameter is regarded as the outer diameter) is, for example, about 0.1 to about 2000 ⁇ m, about 0.2 ⁇ m to about 2000 ⁇ m, and about 1 to. The range is about 1000 ⁇ m, about 2 to about 500 ⁇ m, about 2 to about 200 ⁇ m, and the like.
  • the diameter of the core layer of the multilayer polymer coated crosslinked alginate gel fiber is, for example, about 0.1 to about 2000 ⁇ m, about 0.2 ⁇ m to about 2000 ⁇ m, about 1 to about 1000 ⁇ m, about 2 to about 500 ⁇ m, about 2 to about 200 ⁇ m, etc. Is the range of.
  • the diameter of the core layer is preferably less than the diameter of the fiber and preferably 50% or more.
  • the thickness of the cationic polymer layer (b) of the multilayer polymer-coated crosslinked alginate gel fiber can be determined by the method described in "6. Polymer-coated crosslinked alginate gel fiber" described above.
  • the thickness of the anionic polymer layer is, for example, about 0.1 to about 200 ⁇ m, about 1 to about 200 ⁇ m, about 5 ⁇ m to about 200 ⁇ m, and the like.
  • the values of the core layer diameter, outer diameter, inner diameter of the cationic polymer layer, and inner diameter of the anionic polymer layer of the above-mentioned multilayer polymer-coated crosslinked alginate gel fiber are determined, for example, by using a polymer that develops a fluorescent color in each polymer layer. It can be manufactured and measured from an image taken with a phase-difference optical microscope. It is expressed as the average value of the measured values at several points of the multilayer polymer coated crosslinked alginate gel fiber.
  • the core layer and polymer layer of the above multilayer polymer coated crosslinked alginate gel fiber usually have a substantially uniform thickness, preferably each layer has a thickness uniformity within the range of ⁇ 10%. ..
  • the length of the multilayer polymer coated crosslinked alginate gel fiber is not particularly limited, but is, for example, about 0.01 to about 100 m, or about 0.1 to about 75 m, or about 0.3 to about 50 m. be.
  • the core layer of the multilayer polymer-coated crosslinked alginate gel fiber of some embodiments contains cells capable of producing an antibody, a physiologically active substance, or the like, and is a chemistry represented by the formulas (I) and (II). It can be formed using a mixed solution of modified alginic acid derivatives. In that case, the concentration range of the solution of the chemically modified alginic acid derivative represented by the formula (I) or the formula (II) is the above-mentioned 6. Same as described for polymer coated crosslinked alginate gel fibers.
  • chemically modified alginic acid represented by the formulas (I) and (II) containing cells capable of producing an antibody, a physiologically active substance, etc. used for forming a core layer of a multilayer polymer-coated crosslinked alginic acid gel fiber.
  • concentration of the mixed solution containing the chemically modified alginic acid derivative represented by the formulas (I) and (II) the concentration of the alginic acid solution, the combination of concentrations, and the volume of each solution.
  • the ratio is the same as that described in "6. Polymer-coated crosslinked alginate gel fiber" described above.
  • the solvent used for preparing the solution of the chemically modified alginic acid derivative represented by the formula (I) or the formula (II), the alginic acid solution, etc. used for producing the core layer of the multilayer polymer-coated crosslinked alginic acid gel fiber is described above. It is the same as the solvent described in "6. Polymer-coated crosslinked alginate gel fiber".
  • Anionic polymer A polyanion is a compound having two or more anionic groups in one molecule, and an anionic group is an anionic group or a group that can be induced by an anionic group.
  • the anionic group include acidic functional groups such as a carboxyl group and a sulfuric acid group.
  • the anionic polymer layer is a layer that covers the outside of the cationic polymer layer by electrostatic interaction with the cationic polymer.
  • the anionic polymer forming the anionic polymer layer is not particularly limited as long as it can form a film by electrostatic interaction with the cationic polymer, but is not particularly limited, and is, for example, an acidic functional group such as a carboxyl group or a sulfuric acid group. Examples thereof include polymers having.
  • polysaccharides such as alginic acid, polygalacturonic acid, pectin, pectinic acid, carboxymethyl cellulose, carrageenan, hyaluronic acid and chondroitin sulfate
  • synthetic polymers such as polyacrylic acid and polystyrene sulfonic acid, and their chemistry. Modified products are mentioned, and these chemically crosslinked products are also included.
  • the anionic polymer may have a property of being able to form a film by electrostatic interaction with the cationic polymer, and may be a polymer showing neutrality as a whole.
  • the anionic polymer is a substance capable of coating the cationic polymer layer of the polymer-coated crosslinked alginate gel fiber as an anionic polymer layer and forming a multilayer polymer-coated crosslinked alginate gel fiber (see FIG. 9). It is preferable to have.
  • the anionic polymer is an anion in which an antibody, a physiologically active substance, or the like produced in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber permeates the cationic polymer layer and then coats the cationic polymer layer. It is preferably a substance that can permeate the sex polymer layer and be released out of the fiber.
  • the anionic polymer layer is a group consisting of, for example, anionic polysaccharides, sulfated polysaccharides, synthetic polymers, anionic polyamino acids, chemically modified products thereof, crosslinked products thereof, mixtures thereof, and the like.
  • Anionic polymer selected from, specifically, anionic such as alginic acid, hyaluronic acid, polysaccharideuronic acid, carrageenan, succinoglucan, arabic rubber, xanthan gum, pectin, pectic acid, carboxymethyl cellulose, agar and the like.
  • the anionic polymer layer is, for example, alginic acid, a chemically modified alginic acid derivative represented by the above formula (I), a chemically modified alginic acid derivative represented by the formula (II), and a chemically modified formula (I).
  • Anionic polyamino acids such as polyaspartic acid, chemically modified products thereof, crosslinked products thereof, mixtures thereof, and the like, which are anionic polymers selected from the group.
  • the anionic polymer layer is preferably alginic acid, a chemically modified alginic acid derivative represented by the above formula (I), a chemically modified alginic acid derivative represented by the formula (II), and a chemistry represented by the formula (I).
  • An anionic polymer selected from the group consisting of a crosslinked product formed from a modified alginic acid derivative and / or a chemically modified alginic acid derivative represented by the formula (II), and a mixture thereof.
  • the solvent for preparing the solution containing the anionic polymer is not particularly limited as long as it can dissolve the anionic polymer, but for example, water (tap water, pure water (for example, distilled water, ion-exchanged water, RO). Water, RO-EDI water, etc.), ultrapure water (MilliQ water)), aqueous solution of inorganic salts (phosphate buffered physiological saline (PBS), physiological saline, etc.), and the like, preferably water or Physiological saline is used.
  • water tap water, pure water (for example, distilled water, ion-exchanged water, RO). Water, RO-EDI water, etc.), ultrapure water (MilliQ water)
  • aqueous solution of inorganic salts phosphate buffered physiological saline (PBS), physiological saline, etc.
  • PBS phosphate buffered physiological saline
  • physiological saline preferably water or Physiological saline
  • alginic acid a chemically modified alginic acid derivative represented by the formula (I) or the formula (II) is used as an anionic polymer
  • a solution containing a polyvalent (divalent, trivalent) metal ion that causes gelation Excludes.
  • the weight average molecular weight (Mw) of the anionic polymer is not particularly limited, but is, for example, in the range of about 500 to about 5,000,000, and in the range of about 300,000 to about 2,000,000. It ranges from about 150,000 to about 2.5 million and from about 100,000 to about 3,000,000.
  • the weight average molecular weight (Mw) of the anionic polymer can be measured by gel permeation chromatography (GPC).
  • alginic acid when used as the anionic polymer, its weight average molecular weight (Mw) is, for example, in the range of about 150,000 Da to about 2.5 million Da, and from about 150,000 Da to about 800,000 Da. Range, range from about 300,000 Da to about 700,000 Da, about 700,000 Da to about 1,400,000 Da, about 800,000 Da to about 1,500,000 Da, about 1,400,000 to about 2,000, It is in the range of 000 Da, about 1.5 million to about 2.5 million, and the like.
  • Mw weight average molecular weight
  • the weight average molecular weight (Mw) thereof is, for example, about 100,000 to about 3,.
  • the range is in the range of ,000,000, in the range of about 300,000 Da to about 2,500,000, in the range of about 500,000 to about 2,000,000, and the like.
  • the concentration of the solution containing the anionic polymer is not particularly limited, but may be any concentration as long as it can uniformly coat the surface of the cationic polymer layer of the polymer-coated crosslinked alginate gel fiber, for example, about 0.01 to about. Concentrations in the range of 5.0% by weight, about 0.05 to about 1.0% by weight, about 0.1 to about 0.5% by weight, and about 0.15 to about 0.4% by weight. It is preferably 0.15% by weight.
  • the concentration of the solution is For example, about 0.01 to about 5.0% by weight, about 0.05 to about 1.0% by weight, about 0.1 to 0.5% by weight, about 0.15 to about 0.4% by weight.
  • the concentration in the range. Further, for example, the concentration is in the range of about 0.05 to about 0.15% by weight, about 0.075 to about 0.2% by weight, and about 0.15 to about 0.4% by weight.
  • the concentration of the solution is, for example, about 0. At concentrations ranging from 0.01 to about 5.0% by weight, about 0.05 to about 1.0% by weight, about 0.1 to about 0.5% by weight, and about 0.15 to about 0.4% by weight. be. Further, for example, the concentration is in the range of about 0.05 to about 0.15% by weight, about 0.075 to about 0.2% by weight, and about 0.15 to about 0.4% by weight.
  • the concentration of the solution is, for example, about 0.01 to about 5.0% by weight, about 0. Concentrations in the range of 05 to about 1.0% by weight, about 0.1 to about 0.5% by weight, and about 0.15 to about 0.4% by weight. Further, for example, the concentration is in the range of about 0.05 to about 0.15% by weight, about 0.075 to about 0.2% by weight, and about 0.15 to about 0.4% by weight.
  • a mixed solution containing a chemically modified alginic acid derivative represented by the formulas (I) and (II) is used for forming an anionic polymer layer of a multilayer polymer-coated crosslinked alginate gel fiber
  • a mixed solution thereof is used.
  • the concentration of is, for example, about 0.01 to about 5.0% by weight, about 0.05 to about 1.0% by weight, about 0.1 to about 0.5% by weight, and about 0.15 to about 0. Concentrations in the range of 4% by weight. It is preferably 0.15% by weight.
  • a mixed solution containing a chemically modified alginic acid derivative represented by the formulas (I) and (II) and alginic acid is used for forming an anionic polymer layer of a multilayer polymer-coated crosslinked alginic acid gel fiber, the mixture thereof.
  • the concentration of the solution is, for example, about 0.01 to about 5.0% by weight, about 0.05 to about 1.0% by weight, about 0.1 to about 0.5% by weight, about 0.15 to about 0.
  • the concentration is in the range of 0.4% by weight. It is preferably 0.15% by weight.
  • the mixed solution containing a chemically modified alginic acid derivative represented by the formulas (I) and (II) is used for forming an anionic polymer layer of a multilayer polymer-coated crosslinked alginic acid gel fiber
  • the mixed solution thereof is used.
  • a mixed solution containing a chemically modified alginic acid derivative represented by the formulas (I) and (II) and alginic acid is used for forming an anionic polymer layer of a multilayer polymer-coated crosslinked alginic acid gel fiber
  • a mixture thereof is used.
  • Chemically modified alginic acid derivatives represented by the formulas (I) and (II) when used for forming the anionic polymer layer of the multilayer polymer coated crosslinked alginate gel fiber, in the anionic polymer layer after coating. , Chemically modified alginic acid derivatives represented by the formulas (I) and (II) may form chemical crosslinks.
  • the chemical cross-linking is based on the following formula (III-LA): [In formula (III-LA), -CONH- and -NHCO- at both ends represent amide bonds via any carboxyl group of alginic acid; -L 1A- , -L 2A- , and -X A- , The corresponding -L 1- , -L 2- and -X- in the above-mentioned embodiment [1-12] are the same as the definition].
  • the cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are not particularly limited, and are not particularly limited, and are, for example, antibodies (human antibody, humanized antibody). , Chimera antibody, various monoclonal antibodies such as mouse antibody or their bispecific antibody, low molecular weight antibody, various modified antibodies such as sugar chain modified antibody) producing cells, physiologically active substances (enzymes, cytokines, hormones, blood) Examples thereof include cells capable of producing various useful substances useful as coagulation factors, vaccines, etc.) producing cells, pharmaceutical raw materials, chemical raw materials, food raw materials, and the like. Preferably, it is an antibody-producing cell or a physiologically active substance-producing cell.
  • the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are hybridomas (antibody-producing hybridomas) obtained from B cells that produce antibodies, or antibodies. Examples thereof include cultured cells (antibody-producing gene recombinant cells) transformed with an expression vector.
  • the physiologically active substance-producing cells that can be encapsulated in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are cultured cells transformed with a physiologically active substance expression vector (physiologically active substance production). Genetically modified cells).
  • the cultured cells used as a host for these gene recombination are not particularly limited, and examples thereof include microorganisms such as bacteria and yeast, plant cells, insect cells, and animal cells.
  • microorganism used as a host examples include Escherichia coli, budding yeast, fission yeast, Pichia yeast and the like, and examples of insect cells used as a host include Sf9 cells, Sf21 cells, High five cells and the like.
  • Animal cells used as a host include, for example, CHO cells, CHO cell substrains (CHO-K1 cells, CHO-DG44 cells, CHO-DXB11 cells, CHO cells transformed to modify sugar chains, etc.). , COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, PERC6 cells, YB2 / 0 cells, YE2 / 0 cells, 1R983F cells, Namalwa cells, Will-2 cells, Jurkat cells, Vero cells, Molt-4 cells, HEK293 cells, BHK cells, HT-1080 cells, KGH6 cells, P3X63Ag8.653 cells, C127 cells, JC cells, LA7 cells, ZR-45-30 cells, hTERT cells, NM2C5 cells, UACC-812 cells, etc.
  • CHO cells CHO cell substrains (CHO-K1 cells, CHO-DG44 cells, CHO-DXB11 cells, CHO cells transformed to modify sugar chains
  • the cells can be appropriately selected from (some of which are described in the ATCC cell lineage catalog available from American Type Culture Collection). Unless otherwise specified, in the present specification, the term "CHO cell” means a cell including "CHO cell substrain", and other cells also include cells including each cell substrain. means.
  • the antibody-producing cells that can be encapsulated in the core layer of the polymer-coated crosslinked alginate gel fiber and the multilayer polymer-coated crosslinked alginate gel fiber are preferably animal cells transformed with an antibody expression vector, that is, an antibody. Is a transgenic animal cell that produces.
  • the physiologically active substance-producing cell that can be encapsulated in the core layer is preferably an animal cell transformed with a physiologically active substance expression vector, that is, a recombinant animal cell that produces a physiologically active substance.
  • animal cells used as their hosts include CHO cells, CHO cell substrains, COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, or PERC6 cells, HEK293 cells, BHK cells, and HT-. 1080 cells, or C127 cells; more preferably cells selected from the group consisting of CHO cells, CHO cell substrains, Sp2 / 0 cells, and NS0 cells, HEK293 cells, and BHK cells; more preferably. , CHO cells or CHO cell substrains.
  • the host cell for the antibody-producing cell is preferably a CHO cell, a CHO cell substrain, a Sp2 / 0 cell, or an NS0 cell; more preferably a CHO cell or a CHO cell substrain. ..
  • the host cell of the physiologically active substance-producing cell is preferably a CHO cell, a CHO cell substrain, a HEK293 cell, or a BHK cell; more preferably a CHO cell or a CHO cell substrain.
  • the antibody-producing cells that can be contained in the core layer of the polymer-coated crosslinked alginate gel fiber and the multilayer polymer-coated crosslinked alginate gel fiber are not particularly limited, but are used as biopharmaceuticals or biopharmaceutical raw materials. Examples include cells that produce the antibodies that are used.
  • the cells that produce a bioactive substance are not particularly limited, and examples thereof include cells that produce a biopharmacy or a biopharmaceutical raw material.
  • Biopharmaceuticals include, for example, various cancers, autoimmune and inflammatory diseases, eye diseases, blood diseases, neurological diseases, hereditary rare diseases, endocrine and metabolic diseases, cardiovascular diseases, respiratory diseases, digestive diseases, etc.
  • Examples include pharmaceuticals for various diseases such as skin diseases, muscle / bone diseases, and infectious diseases.
  • the specific target of the antibody drug is not particularly limited, but C5 (complement), CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD52, CD79, IL-1 ⁇ , IL-4R, IL-5, IL-6, IL-6R, IL-12, IL-17, IL-17R, IL-23, IFNAR, PCSK9, CGRP, CGRPR, GD2 (ganglioside), HER2 , HER3, TROP2, BCMA, PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, KIR, SLAMF7, RANKL, TNF- ⁇ , BLyS, EGFR, VEGF, VEGFR, FGF, Nectin , Integrin, EpCAM, CCR4, TfR, TF, FIXa, FX, GPVI, sclerostin, amyloid ⁇ , IgE, various viruses, etc.
  • the antibody-producing cells that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are not particularly limited, but specifically, Muromonab-CD3.
  • Idalcizumab Idalcizumab, brinatumomab, brolcizumab, absiximab, couplersizumab, sertrizumab and the like, and the like, cells producing low molecular weight antibodies consisting of antibody fragments.
  • the antibody-producing cells that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are not particularly limited, but specifically, antibody-producing animals.
  • Examples thereof include antibody-producing CHO cells, antibody-producing Sp2 / 0 cells or antibody-producing NS0 cells, and more preferably antibody-producing CHO cells.
  • the antibody-producing animal cells are not particularly limited, but for example, muromonab-CD3-producing CHO cells, trussumab-producing CHO cells, rituximab-producing CHO cells, paribizmab-producing NS0 cells, and paribizmab-producing CHO cells.
  • Infliximab-producing Sp2 / 0 cells Infliximab-producing CHO cells, Baciliximab-producing Sp2 / 0 cells, Baciliximab-producing CHO cells, Tosirizumab-producing CHO cells, Bebashizumab-producing CHO cells, Adalimmab-producing CHO cells, Setuximab-producing Sp2 / 0 cells, CHO cells, omalizumab-producing CHO cells, eculizumab-producing NS0 cells, eculizumab-producing CHO cells, panitzummab-producing CHO cells, ustequinumab-producing Sp2 / 0 cells, ustequinumab-producing CHO cells, gorimumab-producing Sp2 / 0 cells, golimumab-producing CHO cells, canaquinu / 0 cells, canaquinumab-producing CHO cells, denosumab-producing CHO cells
  • the antibody-producing CHO cells include muromonab-CD3-producing CHO cells, trastuzumab-producing CHO cells, rituximab-producing CHO cells, paribizmab-producing CHO cells, infliximab-producing CHO cells, baciliximab-producing CHO cells, tosirizumab-producing CHO cells, and gemtuzumab-producing CHO cells.
  • Cells bebashizumab-producing CHO cells, ibritsumomab-producing CHO cells, adalimumab-producing CHO cells, setuximab-producing CHO cells, ranibizmab-producing CHO cells, omalizumab-producing CHO cells, eclyzumab-producing CHO cells, panitzummab-producing CHO cells, ustequinumab-producing CHO cells, ustequinumab-producing CHO cells Cells, canaquinumab-producing CHO cells, denosumab-producing CHO cells, mogamurizumab-producing CHO cells, sertrizumab-producing CHO cells, ofatumumab-producing CHO cells, pertzzumab-producing CHO cells, brentuccimab-producing CHO cells, natalyzumab-producing CHO cells, nibolumab-producing CHO cells, Producing CHO cells, sekkinumab-producing CHO cells,
  • Examples thereof include cells, roncastuximab-producing CHO cells, thisotumab-producing CHO cells, rice bilizumab-producing CHO cells, brinatumomab-producing CHO cells, brolcizumab-producing CHO cells, absiximab-producing CHO cells, coupler ciszumab-producing CHO cells, and anti-GPVI antibody-producing CHO cells. ;
  • CHO cells selected from the group consisting of trussumab-producing CHO cells, rituximab-producing CHO cells, infliximab-producing CHO cells, tosirizumab-producing CHO cells, adalimumab-producing CHO cells, nivormab-producing CHO cells, and anti-GPVI antibody-producing CHO cells.
  • tosirizumab-producing CHO cells selected from the group consisting of trussumab-producing CHO cells, rituximab-producing CHO cells, infliximab-producing CHO cells, tosirizumab-producing CHO cells, adalimumab-producing CHO cells, nivormab-producing CHO cells, and anti-GPVI antibody-producing CHO cells.
  • tosirizumab-producing CHO cells for example, tosirizumab-producing CHO cells.
  • the antibody thus produced can be modified or modified after production, and specific examples thereof include PEGylation, drug binding modification, and radiolabeling. That is, in the production of modified antibodies such as PEGylated antibodies and antibody-drug conjugates, cells used for producing the raw material antibody (raw material antibody-producing cells) can be mentioned as cells that can be encapsulated in the core layer.
  • the raw material antibody-producing cell is not particularly limited, and examples of the raw material antibody-producing cell for the PEGylated antibody include cells that produce a raw material antibody fragment of celltrizumab pegol, specifically, celltrizumab-producing CHO cells and the like.
  • the raw material antibody-producing cells of the antibody-drug complex for example, gemtuzumab ozogamycin, ibritsumomab chiuxetan, trusszumab emtancin, trusszumab derkustecan, brentuximab vedotin, inotsumab ozogamycin, inotsumob ozobamycin, setuximab salotalobin.
  • Examples thereof include cells producing raw material antibodies such as verantamab mahodotin, roncastuximab tesillin, thisotumab vedotin, datpotamab derkustecan, patritumab derkustecan, and specific examples thereof include gemtuzumab-producing NS0 cells, ibritsumomab-producing CHO cells, and trussumab-producing CHO cells.
  • Tuximab-producing CHO cells inotsumab-producing CHO cells, setuximab-producing Sp2 / 0 cells, poratuzumab-producing CHO cells, enholtumab-producing CHO cells, sashitsuzumab-producing CHO cells, verantamab-producing CHO cells, Roncus tsuximab-producing CHO cells, chisotumab-producing CHO cells and the like can be mentioned.
  • cells producing a fusion protein of an antibody or antibody fragment and another protein or peptide can also be contained in the core layer, and examples thereof include pavinafspalpha-producing CHO cells and bintrafuspalpha-producing CHO cells. Be done.
  • Antibody will be described in detail in "14. Classification of antibody” and “15. Production and purification method of antibody / physiologically active substance”.
  • the physiologically active substance means a substance and a group of compounds that exert a physiological action or a pharmacological action on an organism.
  • Substances and compound groups that exhibit physiological and pharmacological effects on living organisms include, for example, enzymes, insulin, alkaloids, cytokines (interferon, interleukin, chemokine, tumor necrosis factor, etc.), plant hormones, neurotransmitters, and pheromones. , Hormones (animal hormones), growth factors, growth regulators, growth inhibitors, activators, hematopoietic factors, blood coagulation factors, vaccines (attenuated vaccines, inactivated vaccines, protein vaccines, etc.) and the like.
  • receptors for these substances are also substances that exhibit physiological and pharmacological actions, and are included in physiologically active substances.
  • substances that modify or modify the bioactive substances in addition to the bioactive substances originally possessed by the living body, substances that modify or modify the bioactive substances, substances that activate or inhibit the bioactivity, multiple bioactive substances or fusion proteins that combine some regions or fragments thereof.
  • the physiologically active substance is preferably a proteinaceous physiologically active substance, that is, a physiologically active substance composed of a protein or a peptide.
  • the physiologically active substance-producing cells that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are not particularly limited, but as described above, they are biopharmaceuticals. Alternatively, cells producing a physiologically active substance used as a raw material for biopharmaceuticals can be mentioned.
  • the physiologically active substance used as a biopharmaceutical is not particularly limited, but for example, t-PA, glucocerebrosidase, galactosidase, hyaluronidase, islonidase, glucosidase, sulfatase, uric acid oxidase, DNA degrading enzyme, adenosindeaminase, etc.
  • Enzymes such as trypeptidylpeptidase, hyaluronidase, phenylalanine ammonia lyase, alkaline phosphatase; blood coagulation factors such as FVIIa, FVIII, FIX, FXIII, thrombomodulin, antithrombin, albumin and blood-related proteins; insulin, growth hormone, diuretic peptide, gonads Stimulator hormones, GLP-1, GLP-2, parathyroid hormones, leptin and other hormones; interferons such as IFN- ⁇ , IFN- ⁇ , IFN- ⁇ ; hematopoietic factors such as erythropoetin and thrombopoetin; G-CSF, IL-2 , IL-10, IL-2R, IL-4R, IL-5R, IL-6R, IL-17R, TNFR, EGF, EGFR, FGF, VEGF, VEGFR, PDGF, PDGFR
  • Receptors Cell surface antigens such as CTLA-4, cell surface receptors and their ligands; vaccines such as hepatitis B virus-derived antigens, papillomavirus-derived antigens, varicella-zhelvous virus-derived antigens, and SARS-CoV-2-derived antigens. Examples thereof include proteins and peptides for use, and these subtypes, subunits, and active fragments are also mentioned, and cells producing these physiologically active substances can be contained in the core layer.
  • the physiologically active substance includes a substance having a structural modification, and examples thereof include a substance having an amino acid sequence modification that changes the activity of the substance, and specific examples thereof include insulin analogs and GLP.
  • a substance having an amino acid sequence modification that changes the activity of the substance and specific examples thereof include insulin analogs and GLP.
  • insulin analogs and GLP include insulin analogs and GLP.
  • -1 Analog, erythropoetin analog, etc. may be mentioned. It also includes a substance consisting of the amino acid sequences of some regions or fragments of the original substance, and may be a substance in which a plurality of amino acid sequences of those partial regions or fragments are combined; specifically, insulin analogs. Examples include FVIII analogs and parathyroid hormone analogs.
  • fusion proteins that combine two or more substances and some regions or fragments thereof are also included, for example, fusion proteins of enzymes and antibodies, fusion proteins of cytokine receptors and antibody Fc moieties, cell surface antigen extracellular domains. And the fusion protein of the antibody Fc portion, the fusion protein of the blood coagulation system factor and the antibody Fc portion, the fusion protein of the blood coagulation system factor and the plasma protein, and the like.
  • the core layer can contain cells that produce these structurally modified bioactive substances.
  • the physiologically active substance thus produced can be modified or modified after production, and specific examples thereof include PEGylation, sugar chain modification, drug binding modification, and radiolabeling. That is, it can be used for the production of a protein / peptide as a raw material in the production of modified proteins / peptides such as PEGylated protein and fatty acid-added peptide.
  • modified proteins / peptides such as PEGylated protein and fatty acid-added peptide.
  • PEGylated FVIII, PEGylated erythropoetin, and fatty acid-added insulin analog examples thereof include cells that produce raw material proteins / peptides such as, and cells that produce physiologically active substances as raw materials (raw material physiologically active substance-producing cells) can be included in the core layer.
  • the physiologically active substance-producing cells that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are not particularly limited, but specifically, interferonase. , Monteplase, imiglucerase, veraglucerase, agarcidase, laronidase, alglucosidase, avalglucosidase, idursulfase, galsulfase, erosulfase, lasbricase, dornase, cellliponase, glucarpidase, hyaluronidase, ashotase, etc.
  • interferonase Monteplase, imiglucerase, veraglucerase, agarcidase, laronidase, alglucosidase, avalglucosidase, idursulfase, galsulfas
  • Octocog Lurioctocog, Turoctocog, Ronoctocog, Damoctocog, Simoctocog, Nonacog, Arbutrepenonacog, Catridecacog, Efraloctocog, Eftrenonacog, Thrombomodulin, Antithrombin, Bonicog, Albumin and other blood coagulation factors Cells; insulin, insulin lispro, insulin asparto, insulin glargine, insulin detemil, insulin glulysine, insulin degludec, somatropin, somapcitan, mechaselmin, carperitide, bosolitide, glucagon, holitropin, coriogonadotropin, duraglutide, coriogonadotropin, duraglutide, lylaglutide, lylaglutide Cells that produce hormones such as; cells that produce interferon such as interferon alpha-2a, interferon alpha-2b, inter
  • the physiologically active substance-producing cells that can be contained in the core layer of the polymer-coated cross-linked alginate gel fiber and the multilayer polymer-coated cross-linked alginate gel fiber are not particularly limited, but specifically, physiological.
  • examples thereof include active substance-producing animal cells, preferably physiologically active substance-producing CHO cells, physiologically active substance-producing HEK293 cells, or physiologically active substance-producing BHK cells, and more preferably physiologically active substance-producing CHO cells.
  • physiologically active substance-producing animal cells are not particularly limited, but for example, alteplase-producing CHO cells, imiglucerase-producing CHO cells, agarsidase-producing CHO cells, laronidase-producing CHO cells, and alglucosidase-producing cells.
  • CHO cells aval glucosidase-producing CHO cells, idulsulfase-producing CHO cells, galsulfase-producing CHO cells, erosulfase-producing CHO cells, dronase-producing CHO cells, cell liponase-producing CHO cells, hyaluronidase-producing CHO cells, ashotase-producing CHO cells , Lurioctocog-producing CHO cells, Turoctocog-producing CHO cells, Ronoctocog-producing CHO cells, Nonacog-producing CHO cells, Arbutrepenonacog-producing CHO cells, Thrombomodulin-producing CHO cells, Anti-thrombin-producing CHO cells, Bonikog-producing CHO cells, Holitropin-producing CHO cells, coriogonadotropin-producing CHO cells, duraglutide-producing CHO cells, interferon beta-1a-producing CHO cells, epoetin
  • the physiologically active substance-producing CHO cells include alteplase-producing CHO cells, alglucosidase-producing CHO cells, rulioctocog-producing CHO cells, duraglutide-producing CHO cells, interferon beta-1a-producing CHO cells, darbepoetin-producing CHO cells, and etanelcept-producing CHO cells. , Afribelcept-producing CHO cells, avatarcept-producing CHO cells and the like.
  • the bioactive substances listed here may have the names of substances that have been modified or modified after production as described above, but they are based on the cells that produce the bioactive substances that are the raw materials. Can be included in the layer.
  • PEGylated physiologically active substances such as belfermin
  • cells producing the physiologically active substances as raw materials thereof can be contained in the core layer.
  • cells capable of producing physiologically active substances include natural cells and cells that have been artificially modified, and are cell clusters composed of a plurality of cells. Also included, for example, insulin secretory cells, pancreatic islets, pancreatic islet cells, dopamine secretory cells, pituitary cells, growth hormone secretory cells, parathyroid cells, nerve growth factor secretory cells, blood coagulation factor secretory cells, hepatocytes, small epithelium. Examples thereof include somatic cells, erythropoetin-secreting cells, norepinephrine-secreting cells and the like.
  • the cells producing the physiologically active substance are, in some embodiments, insulin-secreting cells, islets or islet cells, or MIN6 cells derived from pancreatic ⁇ -cells.
  • the "insulin-secreting cell” means a cell having a function of secreting insulin, and for example, in the cells constituting the pancreatic islet, it means a ⁇ cell secreting insulin.
  • the "insulin-secreting cell” may be a cell having an insulin-secreting function due to differentiation, maturation, modification, or the like, and may be, for example, an iPS cell, an ES cell, or a somatic stem cell (for example, a mesenchymal system).
  • Cells having an insulin secretory function obtained by differentiating stem cells such as stem cells) cells having an insulin secretory function obtained by maturing immature cells and precursor cells, and cells having an insulin secretory function obtained by gene recombination. Can also contain cells.
  • the differentiation or maturation of the cell includes culturing the cell, that is, the cell obtained by differentiation or maturation may include the cell obtained by culturing.
  • the “islets”, also known as Langerhans islets, are cell masses composed of an average of about 2000 islet cells. Pancreatic islands are 5 cells that secrete glucagon, ⁇ cells that secrete insulin, ⁇ cells that secrete somatostatin, ⁇ cells that secrete grelin, and PP (pancreatic polypeptide) cells that secrete pancreatic polypeptide. Consists of seed cells.
  • the "islet cell” may be any cell containing at least one of the five types of cells constituting the above-mentioned islets, but preferably contains at least ⁇ cells.
  • the islet cells may be a mixture containing all of ⁇ cells, ⁇ cells, delta cells, ⁇ cells, and PP cells, or may be in a state contained in islets.
  • the “islet cells” may be those that have become islet cells due to differentiation, maturation, modification, or the like.
  • the “pancreatic islet cell” includes, for example, pancreatic islet cells obtained by differentiating stem cells such as iPS cells, ES cells, and somatic stem cells (for example, mesenchymal stem cells), and immature cells and progenitor cells. It may also contain pancreatic islet cells obtained by maturing.
  • Insulin-secreting cells or “islets (including islet cells)" when used for transplantation, have viability and function to the extent that the patient's pathological condition can be recovered when transplanted into the patient. It is preferable to have.
  • Functions of insulin-secreting cells, islets or islets cells include, for example, secreting insulin, and it is preferable that glucose responsiveness is maintained even after transplantation.
  • Donors of "insulin-secreting cells”, “islets” or “islets cells” are animals, preferably vertebrates, more preferably mammals, and specifically include humans, pigs, monkeys, rats or mice. More preferably, it is human or porcine. Donors of "insulin-secreting cells”, “islets” or “islets” are, in some embodiments, pigs in terms of resolving the donor shortage.
  • the "insulin-secreting cell”, “islet” or “islet cell” may be any of pancreatic islets or islet cells obtained from a donor animal, or insulin-secreting cells or pancreatic islet cells obtained from a donor-derived cell. For example, insulin-secreting cells or islet cells differentiated from human-derived ES cells or iPS cells may be used.
  • insulin-secreting cells When “insulin-secreting cells”, “islets” or “islets” are derived from pigs, they are obtained from adult porcine islets, or embryonic, neonatal or perinatal porcine islets, or the islets. Insulin-secreting cells or pancreatic islet cells.
  • the pancreatic islets may be appropriately cultured before use, or pancreatic islets matured from embryonic, neonatal, or perinatal porcine islets may be used.
  • blood coagulation factor secreting cells examples include factor VIII secreting cells and factor IX secreting cells.
  • Method for Producing Polymer-Coated Crosslinked Alginic Acid Gel Fiber a cross-linking reaction is carried out using chemically modified alginic acid derivatives represented by the formulas (I) and (II), which contain cells capable of producing antibodies, physiologically active substances and the like.
  • a method for producing a polymer-coated crosslinked alginate gel fiber in which the crosslinked alginate gel (core layer) formed by the above is coated with a cationic polymer (cationic polymer layer).
  • a method for manufacturing the fiber is provided, which comprises using the apparatus XX shown in FIG.
  • the method for producing the polymer-coated crosslinked alginate gel fiber is not particularly limited, but for example, it is carried out using the apparatus XX shown in FIG.
  • the device XX here is a device preferably used for producing a polymer-coated crosslinked alginate gel fiber.
  • the device XX is, for example, as shown in FIG. 3, a device capable of creating a microchannel having one introduction port and one discharge port, and introduces a solution from the introduction port to an appropriate speed.
  • the solution becomes fibrous (fibrous) and is discharged from the discharge port.
  • the device XX is, for example, a cell capable of producing an antibody, a physiologically active substance, etc. introduced from the introduction port of the device XX using an extrusion tube YY or the like as shown in FIG. 3, and formulas (I) and (II).
  • the mixed solution By extruding the mixed solution containing the chemically modified alginic acid derivative represented by the above, the mixed solution can be ejected from the outlet of the apparatus XX.
  • an injection tube can be used as a device provided with the device XX and the extruder YY.
  • the device XX becomes an outer cylinder
  • the extrusion cylinder YY for extruding the solution introduced into the device XX from the discharge port becomes an inner cylinder.
  • a glass or plastic syringe can be used.
  • a container DD such as a beaker containing a solution containing divalent metal ions is used as a container for receiving the fibrous substance discharged from the discharge port 2 of the apparatus XX.
  • a container EE such as a beaker containing a solution containing the cationic polymer is used as a container for coating the surface of the crosslinked alginate gel fiber CLA with a cationic polymer.
  • FIG. 3 is a schematic diagram illustrating one aspect of the manufacturing process of the polymer-coated crosslinked alginate gel fiber.
  • a production method using a mixed solution of a chemically modified alginic acid derivative represented by the formulas (I) and (II) containing cells (cells capable of producing an antibody, a physiologically active substance, etc.) will be described.
  • the polymer-coated crosslinked alginate gel fiber can be produced, for example, by a method including the following steps (S) to (2).
  • Step (S) A step of introducing a mixed solution containing a cell capable of producing an antibody, a physiologically active substance, etc. and a chemically modified alginic acid derivative represented by the formulas (I) and (II) from the introduction port 1 of the apparatus XX.
  • Step (1) A mixed solution containing cells capable of producing antibodies, physiologically active substances, etc. and chemically modified alginic acid derivatives represented by the formulas (I) and (II) from the outlet 2 of the apparatus XX, is a divalent metal.
  • a solution containing a chemically modified alginic acid derivative represented by the formulas (I) and the formula (II) is prepared for cells capable of producing an antibody, a physiologically active substance and the like described by the cells contained in the core layer described above. Suspended or dissolved in. At this time, in addition to cells capable of producing an antibody, a physiologically active substance, or the like, components such as an alginic acid solution, a medium, a culture solution, a collagen solution, methyl cellulose, and a sucrose solution can be added.
  • step (S) prepared in step (S) and chemically modified alginic acid derivatives represented by the formulas (I) and (II).
  • step (S) chemically modified alginic acid derivatives represented by the formulas (I) and (II).
  • the released solution is sequentially gelled, whereby a fibrous (fibrous) structure can be produced.
  • ionic cross-linking proceeds between the chemically modified alginic acid derivatives represented by the formulas (I) and (II), and at the same time, chemical cross-linking by the Huisgen reaction also proceeds, and a gel is prepared. can.
  • step (2) the crosslinked alginate gel fiber containing the cells capable of producing the antibody, the physiologically active substance, etc.
  • the polymer-coated crosslinked alginate gel fiber (CFB) of the present invention can be produced.
  • contact refers to a solution (eg, a solution of a chemically modified alginic acid derivative) or a gel (eg, a crosslinked alginate gel) to another solution (eg, a solution containing divalent metal ions, a cationic polymer). It means that it is immersed in or added to a solution containing, a solution containing an anionic polymer, and the like.
  • a solution eg, a solution of a chemically modified alginic acid derivative
  • a gel eg, a crosslinked alginate gel
  • another solution eg, a solution containing divalent metal ions, a cationic polymer
  • the flow velocity (injection rate) of the mixed solution containing the antibody, the cell capable of producing the physiologically active substance, etc. ejected from the outlet 2 of the apparatus XX and the chemically modified alginic acid derivative represented by the formulas (I) and (II) is, for example. , About 100 to about 10000 ⁇ L / min.
  • the flow velocity is, for example, 250 ⁇ L / min, 4 mL / min, 10 mL / min, and the like.
  • the flow velocity for producing a polymer-coated crosslinked alginate gel fiber containing MIN6 cells in the core layer is, for example, 125 ⁇ L / min.
  • the flow velocity (injection speed) can be adjusted using a syringe pump or the like, and fibers of various sizes can be manufactured. Further, by changing the size (diameter) of the discharge port 2 of the apparatus XX, it is possible to manufacture a fiber in which the diameter of the core layer can be adjusted.
  • a luer lock needle material such as metal
  • the mixed solution containing the cells capable of producing the antibody, the physiologically active substance, etc. introduced from the introduction port 1 of the apparatus XX and the chemically modified alginic acid derivatives represented by the formulas (I) and (II) is, for example, the above-mentioned embodiment [1].
  • the chemically modified alginic acid derivatives represented by the formulas (I) and (II) described in the above are used in a solvent (for example, medium, cell culture medium, culture solution, isotonic buffer solution, phosphate buffered physiological saline, and A predetermined concentration (for example, the concentration of the solution of each chemically modified alginic acid derivative is about 0.01 to about 1.5% by weight, expressed by the formulas (I) and (II)) by adding physiological saline or the like.
  • concentration of the mixed solution of the chemically modified alginic acid derivative is about 0.02 to about 2.0% by weight), and the mixed solution of the chemically modified alginic acid derivative represented by the formula (I) and the formula (II) is prepared.
  • the alginic acid solution When the alginic acid solution is added to a mixed solution containing a cell capable of producing an antibody, a physiologically active substance, etc. introduced from the introduction port 1 of the apparatus XX and a chemically modified alginic acid derivative represented by the formulas (I) and (II).
  • the total concentration of the mixed solution containing the chemically modified alginic acid derivative represented by the formula (I) and the formula (II) and the concentration of the alginic acid solution is prepared, for example, in the range of about 0.5 to about 2.0% by weight. To.
  • the alginic acid solution When the alginic acid solution is added to the mixed solution containing the cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by the formulas (I) and (II) introduced from the introduction port 1 of the apparatus XX.
  • the combination of the concentration (C1 (% by weight)) of the mixed solution containing the chemically modified alginic acid derivative represented by the formulas (I) and (II) and the concentration of the alginic acid solution (C2 (% by weight)) is not particularly limited.
  • (C1: C2) (about 0.2: about 1.3), (about 0.5: about 1.0), (about 1.0: about 0.5), (about 1.5). : 0), (about 0.66: about 1.34), (about 0.34: about 0.66), (about 0.16: about 0.34) and the like.
  • the concentrations of C1 and C2 can be appropriately combined and prepared.
  • the alginic acid solution When the alginic acid solution is added to the mixed solution containing the cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by the formulas (I) and (II) introduced from the introduction port 1 of the apparatus XX. Concentration of the solution of the chemically modified alginic acid derivative represented by the formula (I) (C1A (% by weight)), the concentration of the solution of the chemically modified alginic acid derivative represented by the formula (II) (C1N (% by weight)), and the concentration of the alginic acid solution.
  • the alginic acid solution When the alginic acid solution is added to the mixed solution containing the cells capable of producing antibodies, physiologically active substances, etc. and the chemically modified alginic acid derivatives represented by the formulas (I) and (II) introduced from the introduction port 1 of the apparatus XX.
  • the volume of the chemically modified alginic acid derivative represented by the formula (I) (v1), the volume of the chemically modified alginic acid derivative represented by the formula (II) (v2), and the volume of the alginic acid solution in the mixed solution to which the alginic acid solution is added (v1).
  • the outer diameter of the produced polymer-coated crosslinked alginate gel fiber CFB is not particularly limited, but is as described above, and is, for example, in the range of about 0.1 to about 200 ⁇ m.
  • the length of the polymer-coated crosslinked alginate gel fiber CFB is not particularly limited and is as described above, and may be, for example, about 0.3 to about 50 m.
  • the cross-sectional shape of the fiber is as described above, and examples thereof include a circle, an ellipse, a polygon such as a quadrangle and a pentagon, and the like.
  • the solution contained is as described in the above-mentioned "5-1.
  • Crosslinked alginate gel and examples thereof include a solution containing calcium ion, magnesium ion, barium ion, strontium ion, zinc ion and the like.
  • the divalent metal ion concentration of the solution containing the divalent metal ion is, for example, in the range of about 1 mM to about 1 M, or about 10 to about 500 mM; preferably about 10 to about 100 mM.
  • the solvent used when preparing the solution containing the divalent metal ion is as described in the above-mentioned "5-1.
  • Crosslinked alginate gel and examples thereof include water and physiological saline.
  • the contact time is, for example, about 1 minute to 60 minutes, 1 minute to 30 minutes, and the like.
  • the solution containing the cationic polymer to which the crosslinked alginate gel fiber CLA obtained in the step (2) of the method for producing the polymer-coated crosslinked alginate gel fiber is brought into contact is the cationicity described in the above-mentioned "7. Cationic polymer”. It is a solution containing a polymer, and examples thereof include a solution containing a polyamino acid, a basic polysaccharide, a basic polymer and the like.
  • the concentration of the solution containing the cationic polymer to be contacted with the crosslinked alginate gel fiber CLA is as described in "7. Cationic polymer" described above, for example, from about 0.02 to about 0.2% by weight. , About 0.05 to about 0.1% by weight, etc.
  • the solution containing the cationic polymer to which the crosslinked alginate gel fiber (CLA) is contacted is an aqueous solution of calcium chloride, an aqueous solution of sodium chloride, and a buffer solution for adjusting the pH of the solution (acetic acid, sodium acetate, sodium hydroxide, hydroxyethylpiperazine). It can contain components such as an aqueous solution of ethanesulfonic acid).
  • the time for contacting the crosslinked alginate gel fiber CLA with the solution containing the cationic polymer is, for example, about 1 minute to 60 minutes, 1 minute to 30 minutes, or the like.
  • the temperature during the process of producing the polymer coated crosslinked alginate gel fiber is, for example, in the range of about 4 to about 37 ° C.
  • a polymer-coated crosslinked alginate gel fiber having a core layer containing a certain number of cells capable of producing an antibody, a physiologically active substance, or the like can be easily obtained.
  • antibody-producing cells, physiologically active substance-producing cells, and the like can be cultured to produce antibodies, physiologically active substances, and the like.
  • the polymer-coated crosslinked alginate gel fiber can be continuously cultured for several weeks to several months, such as antibody-producing cells and physiologically active substance-producing cells, by appropriately exchanging the culture solution.
  • the strength of the polymer-coated crosslinked alginate gel fiber can be measured by a shaking disintegration test, a tensile strength test, or the like according to a method well known to those skilled in the art.
  • Method for Producing Multilayer Polymer-Coated Crosslinked Alginic Acid Gel Fiber a cross-linking reaction is carried out using chemically modified alginic acid derivatives represented by the formulas (I) and (II), which contain cells capable of producing antibodies, physiologically active substances and the like.
  • a method for producing a multilayer polymer coated crosslinked alginate gel fiber in which the crosslinked alginate gel (core layer) formed thereby is coated with a cationic polymer (cationic polymer layer) and an anionic polymer (anionic polymer layer).
  • Ru a method for producing a multilayer polymer coated crosslinked alginate gel fiber using a polymer coated crosslinked alginate gel fiber (CFB) obtained by using the apparatus XX shown in FIG. 3 or FIG. 10 is provided.
  • the multilayer polymer-coated crosslinked alginate gel fiber is manufactured by performing the following step (3) following the steps (S) to (2) described in the above "11. Method for manufacturing a polymer-coated crosslinked alginate gel fiber". can do.
  • Step (3) A step of contacting the polymer-coated crosslinked alginate gel fiber (CFB) obtained in the step (2) with a solution containing an anionic polymer and further coating with the anionic polymer.
  • step (3) the polymer-coated crosslinked alginate gel fiber (CFB) containing cells capable of producing the antibody, physiologically active substance, etc. obtained in step (2) is brought into contact with a solution containing an anionic polymer to obtain an antibody.
  • the surface of the polymer-coated crosslinked alginate gel fiber containing cells capable of producing physiologically active substances and the like is coated with an anionic polymer layer.
  • the multilayer polymer-coated crosslinked alginate gel fiber (ACFB) of the present invention can be produced.
  • a container FF such as a beaker containing a solution containing the anionic polymer shown in FIG. 10 is used.
  • the outer diameter of the produced multilayer polymer-coated crosslinked alginate gel fiber is not particularly limited, but is as described above, and is, for example, in the range of about 0.1 to about 200 ⁇ m.
  • the length of the multilayer polymer-coated crosslinked alginate gel fiber (ACFB) is not particularly limited and is as described above, and may be, for example, about 0.3 to about 50 m.
  • the cross-sectional shape of the fiber is as described above, and examples thereof include a circle, an ellipse, a polygon such as a quadrangle and a pentagon, and the like.
  • the anionic polymer to which the polymer-coated crosslinked alginate gel fiber (CFB) obtained in the step (2) is brought into contact is the anionic polymer described in the above-mentioned "9.
  • Anionic polymer and is preferably anionic poly.
  • Anionic polymers selected from the group consisting of saccharides, sulfated polysaccharides, synthetic polymers, anionic polyamino acids, chemically modified products thereof, crosslinked products thereof, mixtures thereof, and the like.
  • the polymer-coated crosslinked alginic acid gel fiber (CFB) obtained in the step (3) has alginic acid, a chemically modified alginic acid derivative represented by the above formula (I), and a chemically modified alginic acid derivative represented by the formula (II).
  • the surface of the polymer-coated crosslinked alginic acid gel fiber (CFB) coated with the cationic polymer is further coated with alginic acid, the chemically modified alginic acid derivative represented by the above formula (I), and the chemically modified alginic acid represented by the formula (II). It is coated with a derivative or the like.
  • the concentration of the solution containing the anionic polymer to which the polymer coated crosslinked alginate gel fiber (CFB) is contacted is as described in "9. Anionic Polymer" above, for example, from about 0.01 to about 5.
  • the concentration is in the range of 0.0% by weight, about 0.05 to about 1.0% by weight, about 0.1 to about 0.5% by weight, about 0.15 to about 0.4% by weight, and the like.
  • the time for contacting the polymer-coated crosslinked alginate gel fiber (CFB) with the solution containing the anionic polymer is, for example, about 1 minute to 60 minutes, 1 minute to 30 minutes, or the like.
  • the temperature during the process of manufacturing the multilayer polymer coated crosslinked alginate gel fiber is, for example, in the range of about 4 to about 37 ° C.
  • a multilayer polymer-coated crosslinked alginate gel fiber having a core layer containing a certain number of cells capable of producing an antibody, a physiologically active substance, or the like can be easily obtained.
  • multilayer polymer-coated crosslinked alginate gel fiber by culturing the multilayer polymer-coated crosslinked alginate gel fiber in a culture medium, antibody-producing cells, physiologically active substance-producing cells, and the like can be cultured to produce antibodies, physiologically active substances, and the like.
  • the multi-layer polymer-coated cross-linked alginate gel fiber enables continuous culture of antibody-producing cells, physiologically active substance-producing cells, and the like for several weeks to several months by appropriately exchanging the culture medium.
  • the strength of the multilayer polymer-coated crosslinked alginate gel fiber can be measured by a shaking disintegration test, a tensile strength test, or the like according to a method well known to those skilled in the art.
  • a method for producing an antibody, a physiologically active substance, or the like using an alginate gel fiber is provided.
  • the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber is placed in a culture vessel, and a medium is added to impregnate the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber for culturing.
  • a medium is added to impregnate the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber for culturing.
  • the method for culturing antibody-producing cells, physiologically active substance-producing cells, etc. in a preferred embodiment, after producing a polymer-coated crosslinked alginate gel fiber containing cells capable of producing antibodies, physiologically active substances, etc. in the core layer by the above-mentioned production method.
  • the cells can be infiltrated into the culture solution to start culturing antibody-producing cells, physiologically active substance-producing cells, and the like.
  • the culture solution (nutrient source) and oxygen can be immediately supplied to the core layer, that is, antibody-producing cells, bioactive substance-producing cells, etc. contained in the core layer.
  • the antibody, the bioactive substance and the like can be produced while sufficiently preventing necrosis of the antibody-producing cells, the bioactive substance-producing cells and the like in the core layer of the polymer-coated crosslinked alginate gel fiber.
  • a multilayer polymer-coated crosslinked alginate gel fiber containing cells capable of producing antibodies, physiologically active substances, etc. in the core layer was produced by the above-mentioned production method.
  • the cells can be infiltrated into the culture medium to start culturing the antibody-producing cells, the physiologically active substance-producing cells, and the like.
  • the culture solution (nutrient source) and oxygen can be immediately supplied to the core layer of the multilayer polymer-coated crosslinked alginate gel fiber, that is, the antibody contained in the core layer.
  • the antibody, the bioactive substance and the like can be produced while sufficiently preventing necrosis of the antibody-producing cells, the bioactive substance-producing cells and the like in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber.
  • the polymer-coated cross-linked alginate gel fiber or multilayer polymer-coated cross-linked alginate gel fiber containing cells capable of producing the antibody, physiologically active substance, etc. of the present invention in the core layer is a culture solution (nutrient source) existing outside the fiber at the time of culturing. It has sufficient permeability to components such as oxygen.
  • a polymer-coated crosslinked alginate gel fiber having a core layer containing physiologically active substance-producing cells prepared by the above-mentioned production method is placed in a low-adhesion surface dish, and a medium (5 mL) having the composition shown in Table 35 described later is added. Then, incubate in an incubator at 37 ° C. in a 5% CO 2 atmosphere.
  • the culture is started while shaking under the conditions of the above, and after 5 days, the culture temperature is set to 30 ° C., and the culture is continued at the same temperature.
  • the culture period 1.8 mL of the culture medium is withdrawn once every 2 to 3 days, and 1.8 mL of the medium having the composition of Table XX or 1.8 mL of the feed solution (manufactured by Irvine, catalog number JX F003) is added. , Keep the total volume of medium at 30 mL. In addition, half the amount of the culture solution is exchanged once a week.
  • the technique for producing an antibody, a physiologically active substance, etc. using a polymer-coated cross-linked alginate gel fiber or a multilayer polymer-coated cross-linked alginate gel fiber according to a certain aspect includes a certain number of antibody-producing cells, physiologically active substance-producing cells, etc. contained in the core layer. Because it does not proliferate, there is less physical stress on the cells, so there is a possibility that the encapsulated antibody-producing cells, bioactive substance-producing cells, etc. will continue to produce antibodies, bioactive substances, etc. for a long period of time. It is excellent in that it is.
  • the production and purification efficiency of the antibody for example, by using the polymer-coated cross-linked alginate gel fiber or the multilayer polymer-coated cross-linked alginate gel fiber of the preferred embodiment, large-scale culture is performed. Unlike floating culture, which requires a tank, it is possible to cultivate antibodies in a small-scale production facility), as a continuous production technology for next-generation antibody drugs suitable for the production of small-quantity and various types of antibody drugs. You can expect it.
  • the culture-produced antibody eg, anti-GPVI antibody, tosirizumab-producing CHO cells
  • physiologically active substance eg, insulin
  • the culture-produced antibody eg, anti-GPVI antibody, tosirizumab-producing CHO cells
  • physiologically active substance eg, insulin
  • the antibody produced by the culture for example, tosirizumab-producing CHO cells
  • the physiologically active substance may be stored in the core layer of the multi-layer polymer-coated cross-linked alginate gel fiber, preferably the multi-layer polymer-coated cross-linked alginate gel fiber. It permeates the core layer, the cationic polymer layer and the anionic polymer layer and is stored in the culture solution outside the multilayer polymer coated crosslinked alginate gel fiber.
  • antibodies, bioactive substances and the like produced in the core layer of the polymer-coated crosslinked alginate gel fiber permeate the core layer and the cationic polymer layer and are sequentially released out of the fiber. Therefore, it is possible to form a cycle in which continuous culture of antibodies, bioactive substances, etc. is possible. At this time, metabolites and waste products may also be released to the outside of the fiber.
  • an antibody, a physiologically active substance, or the like produced in the core layer of the multilayer polymer-coated crosslinked alginate gel fiber permeates the core layer, the cationic polymer layer, and the anionic polymer layer. It will be sequentially released to the outside of the fiber, and a cycle capable of continuous culture of antibodies, physiologically active substances, etc. can be formed. At this time, metabolites and waste products may also be released to the outside of the fiber.
  • cells selected from anti-GPVI antibody-producing CHO cells, tosirizumab-producing CHO cells, or MIN6 cells are used as cells to be included in the core layer, and the formula used for forming the crosslinked alginate gel in the core layer (
  • a chemically modified alginic acid derivative of I) the following formula:
  • (ALG) represents alginic acid
  • -NHCO- represents an amide bond mediated by any carboxyl group of alginic acid
  • the chemically modified alginic acid derivative selected from the following is the chemically modified alginic acid of the formula (II).
  • (ALG) represents alginic acid; -NHCO- represents an amide bond mediated by any carboxyl group of alginic acid], using a chemically modified alginic acid derivative selected from the above, the cationicity of the cationic polymer layer.
  • Polymer-coated crosslinked alginate gel fibers were made using poly-L-ornithine, polyallylamine (PAA), polyethyleneimine or polymethylene-CO-guanidine (PMCG) as the polymer.
  • the outside of the cationic polymer layer of the polymer-coated crosslinked alginic acid gel fiber produced above is used as an anionic polymer, alginic acid, a chemically modified alginic acid derivative of the following formula (I):
  • (ALG) represents alginic acid; -NHCO- represents an amide bond mediated by any carboxyl group of alginic acid], a chemically modified alginic acid derivative selected from the following formula (II).
  • a crosslinked alginate gel fiber was prepared.
  • the produced antibody anti-GPVI antibody, tosirizumab
  • physiologically active substance insulin
  • Examples of the culture vessel for culturing a polymer-coated cross-linked alginate gel fiber or a multilayer polymer-coated cross-linked alginate gel fiber containing cells capable of producing antibodies, physiologically active substances, etc. in the core layer include a tissue culture plate, a triangular flask, and T-.
  • any method such as static culture, shaking / shaking culture or the like may be selected.
  • the culture temperature is, for example, in the range of about 28 to about 39 ° C., for example, in the range of about 30 ° C. to about 37 ° C. in some methods for producing an antibody, a physiologically active substance, or the like.
  • the culture temperature at the start of culturing is set to about 37 ° C, and the temperature can be changed to about 30 ° C after culturing for a certain period of time.
  • the culture period is, for example, 7 days or longer, 10 days or longer, 20 days or longer, or 30 days or longer. Or 40 days or more, 50 days or more, 60 days or more, or 70 days or more.
  • the culture period is, for example, 7 days, 14 days, 28 days, 35 days, or 42 days. Yes, or 49 days, or 56 days, or 63 days, or 70 days.
  • the cell growth inhibitor is an agent capable of suppressing excessive cell proliferation during the culture period, and for example, addition of dimethyl sulfoxide, sodium butyrate, valproic acid, lithium chloride, valeric acid, methotrexate (MTX) and the like. Agents are mentioned.
  • the timing of adding the cell proliferation inhibitor to the culture solution can be either at the start of the culture or during the culture period (when the cells can grow to the required number of cells). In the present specification, when culturing using anti-GPVI antibody-producing cells, methotrexate (MTX) is added.
  • the cell culture medium a commercially available medium base material, a prepared medium, or a self-made medium can be used. It is also possible to use a natural medium (for example, soybean-casein digest medium (SCD medium) or the like) or a synthetic medium (a medium in which all various nutrients necessary for growth are supplemented with chemicals).
  • the medium is not particularly limited, but may be a basic medium containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, etc.).
  • DMEM Dulbecco's Modified Eagle Medium
  • MEM Minimum Essential Medium
  • BME Basic Medium Eagle
  • Dulbecco's Medium FiberMedium 12 Glassgo Medium Essential Medium
  • G016 medium G016 medium
  • DMEM High Glucose
  • the medium may further contain serum.
  • the serum is not particularly limited, and examples thereof include FBS / FCS (Fetal Bovine / Calf Serum), NCS (Newborn Calf serum), CS (Calf Serum), HS (Horse Serum) and the like.
  • the concentration of serum contained in the medium is, for example, 2% by weight or more and 10% by weight or less.
  • both ends of the crosslinked alginate gel fiber of the core layer of the polymer-coated crosslinked alginate gel fiber of the present invention are coated with a cationic polymer, antibody-producing cells and physiologically active substances contained in the core layer during the culture period are produced. This leads to prevention, suppression or reduction of a large amount of cells such as cells leaking out of the fiber (for example, 1 ⁇ 10 5 or more).
  • both ends of the crosslinked alginate gel fiber of the core layer of the crosslinked alginate gel fiber coated with the multilayer polymer of the present invention are coated with the cationic polymer and the anionic polymer, the antibody-producing cells contained in the core layer during the culture period. This leads to prevention, suppression or reduction of a large amount (for example, 1 ⁇ 10 5 or more) of cells such as physiologically active substance-producing cells leaking out of the fiber. 13-1.
  • Method for calculating the number of viable cells in the core layer The following is included in the core layer of a polymer-coated crosslinked alginate gel fiber or a multilayer polymer-coated crosslinked alginate gel fiber containing antibody-producing cells at the start of culture, during the culture period, or after culture.
  • Cross-linked alginate gel fiber containing antibody-producing cells, polymer-coated cross-linked alginate gel fiber, or multi-layer polymer-coated cross-linked alginate gel fiber (0.2 mL) in a 15 mL tube (esoteric tube (with print scale / bulk), model: 2325-015- Transfer to MYP) and add G016 medium (4.5 mL) having the composition shown in Table 31 described later up to about 4.5 mL on the scale of the tube.
  • antibody Antibodies are referred to as mouse antibodies, rat antibodies, rabbit antibodies, human antibodies and the like, depending on the immune animal species at the time of production.
  • Chimera antibodies and humanized antibodies are modified antibodies obtained by converting partial regions of antibodies derived from different species into human sequences in order to reduce immunogenicity when used in humans, and are used as biopharmacy.
  • an antibody produced from a human antibody gene using a mouse or the like in which a human antibody gene has been incorporated which is also referred to as a human antibody or simply a human antibody and is used as a biopharmacy.
  • next-generation antibodies various modified antibodies called next-generation antibodies have also been developed, and the modified antibodies are also included in the "antibodies" in the present specification.
  • a polyvalent antibody which is an antibody showing specificity to two or more antigens
  • an antibody showing particularly bispecificity is called a bispecific antibody and is one of highly functionalized antibodies.
  • a low-molecular-weight antibody which is an antibody in which the Fc site of the antibody is removed to reduce the molecular weight, and examples thereof include Fab, F (ab') 2 , scFv (single-chain Fv), VHH, etc., which are used as biopharmacy. ..
  • bispecific low molecular weight antibodies have also been produced, for example scFv-scFv being used as biopharmacy.
  • An antibody in which a mutation is added to an Fc region or the like so as to modify a sugar chain is also an example of a modified antibody.
  • a sugar chain-modified antibody can also be produced by pre-transforming a host cell so as to modify the sugar chain, and examples thereof include a fucose-removing antibody.
  • a fusion protein of an antibody or antibody fragment and another protein or peptide is also mentioned as an example of a modified antibody, that is, an antibody, but when it is a fusion protein with the above-mentioned physiologically active substance, it is physiological. It is also contained in active substances.
  • Antibodies are classified into classes (isotypes) and subclasses as shown in the table below, depending on the structural differences in the constant region.
  • the produced antibody is used as a biopharmacy, it is preferably an IgG antibody.
  • the molecular weight of the produced antibody that can permeate the cationic polymer layer and the anionic polymer layer is not particularly limited, but is, for example, an antibody in the range of about 45,000 to about 1,000,000 Da.
  • the molecular weight of the antibody capable of permeating the cationic polymer layer is, for example, about 3,000 to about 1,000,000 Da, about 20,000 to about 1,000,000 Da, and about 20,000 to about 400,000 Da.
  • the molecular weight of the physiologically active substance produced in the core layer of the above and capable of permeating the cationic polymer layer and the anionic polymer layer is not particularly limited, but is, for example, about 3,000 to about 1,000,000 Da.
  • 20,000 to about 1,000,000 Da is a physiologically active substance in the range of 000 Da, about 45,000 to about 200,000 Da.
  • an antibody corresponding to the antibody-producing cells used is produced.
  • muromonab-CD3 producing CHO cells muromonab-CD3 is produced as an antibody.
  • the physiologically active substance corresponding to the used physiologically active substance-producing cells is produced.
  • Examples of the antibody produced include mromonab-CD3 (IgG; 150,000) using mromonab-CD3-producing CHO cells, trusszumab (IgG; 148,000) using trussumab-producing CHO cells, and rituximab-producing CHO cells.
  • paribismab using paribismab-producing NS0 cells (IgG; 147,700), infliximab-producing Sp2 / 0 cells or infliximab-producing CHO cells using infliximab (IgG; 149,000), Baciliximab (IgG; 147,000) using basiliximab-producing Sp2 / 0 cells, tosirizumab (IgG; 148,000) using tosirizumab-producing CHO cells, gemtuzumab (IgG; 150,000) using gemtuzumab-producing CHO cells, Bebasizumab (IgG; 149,000) using bevasizumab-producing CHO cells, ibritsumomab (IgG; 148,000) using ibritsumomab-producing CHO cells, adalimumab (IgG; 148,000) using ad
  • Ecrizumab (IgG; 145,235) using Ecrizumab-producing NS0 cells
  • Panitumumab IgG; 147,000
  • Ustekinumab IgG; 148,079-
  • Ustekinumab-producing Sp2 / 0 cells Ustekinumab
  • golimumab IgG; 149,802 to 151,064
  • canaquinumab IgG; 148,000
  • canaquinumab-producing Sp2 / 0 cells denosumab-producing CHO cells.
  • Denosumab (IgG; 150,000), mogamurizumab (IgG; 149,000) using mogamurizumab-producing CHO cells, sertrizumab (IgG (Fab'); 50,000) using sertrizumab-producing CHO cells, OFATUMMB-producing NS0.
  • Ofatumumab (IgG; 149,000) using cells, peltuzumab (IgG; 148,000) using pertuzumab-producing CHO cells, blurring using lentuximab-producing CHO cells Ntuximab (IgG; 148,000), Natalizumab using Natalizumab-producing NS0 cells (IgG; 146,178), Nivolumab (IgG; 145,000) using nibolumab-producing CHO cells, Alemtuzumab using alemtuzumab-producing CHO cells (IgG; 145,000).
  • IgG; 150,000 sekkinumab (IgG; 151,000) using sekkinumab-producing CHO cells, ramsirumab (IgG; 147,000) using ramsylumab-producing NS0 cells, and ipilimumab (IgG; IgG;) using ipilimumab-producing CHO cells. 148,000), Eborokumab (IgG; 141,789) using Eborokumab-producing CHO cells, Mepolizumab (IgG; 149,000) using Mepolizumab-producing CHO cells, Arilokumab (IgG; 145892.) Using Allirokumab-producing CHO cells. 049.
  • Ixekizumab (IgG; 149,000) using Ixekizumab-producing CHO cells, Brodalmab (IgG; 147,000) using Brodalmab-producing CHO cells, and Idalcizumab (IgG (Fab); 47, using Idalsizumab-producing CHO cells. 782), erotuzumab (IgG; 148,000) using erotuzumab-producing NS0 cells, pembrolizumab (IgG; 149,000) using pembrolizumab-producing CHO cells, and salilumab (IgG; 150,000) using salilumab-producing CHO cells.
  • Bezlotoxmab (IgG; 148,000) using bezlotoxmab-producing CHO cells, berimumab (IgG; 147,000) using berimumab-producing NS0 cells, daratumumab (IgG; 148,000) using daratumumab-producing CHO cells, abelumab.
  • Avelumab (IgG; 147,000) using CHO cells producing, dupilumab (IgG; 152,000) using CHO cells producing dupyrumab, atezolizumab (IgG; 144,611) using CHO cells producing atezolizumab, CHOs producing benlarizumab.
  • Benralizumab (IgG; 148,000) using cells, inotzumab (IgG; 149000) using inotsumab-producing CHO cells, emicizumab (IgG; 148,000) using emicizumab-producing CHO cells, and guselkumab-producing CHO cells.
  • Gesselkmab (IgG; 146,000), Durvalumab (IgG; 149,000) using Durvalumab-producing CHO cells, Obinutsumab (IgG; 148,000-150,000) using Obinutsumab-producing CHO cells, Bedrizumab-producing CHO.
  • the antibody that can be obtained by the method for producing an antibody of the present invention is not particularly limited to the above-mentioned class (isotype) or subclass.
  • the produced antibody is purified, for example, through the following three steps.
  • Step 1 In order to substantially remove proteins and solids other than antibodies contained in the medium, centrifugation or filtration with a filter is performed.
  • Step 2 For example, the target antibody is taken out by chromatography such as affinity chromatography (in the case of an antibody, affinity chromatography using Protein A or Protein G) or ion exchange chromatography.
  • Step 3 In order to remove the contaminants mixed in the step 2, ion exchange chromatography, gel filtration chromatography, hydroxyapatite chromatography and the like are performed to purify the target antibody with high purity.
  • the produced physiologically active substance is purified, for example, by the same method as in the above step.
  • Affinity chromatography with Protein A or Protein G As a method for purifying IgG, for example, a method for purifying an antibody using Protein A or Protein G is known. As an example of the antibody purification method using Protein A, the following method can be mentioned. (1) Using a column filled with beads to which Protein A is fixed, IgG is added to the beads in the column by filtering the solution obtained by adding serum to the solution obtained by the method of the above [step 1]. And other serum components are spilled out of the column. (2) After that, by passing an acidic solution through the column, the IgG bound to the beads is cut off and eluted to the outside of the column to obtain IgG. Since the binding force of Ig to Protein A and Protein G differs depending on the animal species and subclass, Protein A or Protein G can be used properly depending on the purpose.
  • Ion exchange chromatography It is a method of separating proteins by utilizing the electrical properties (charges) of proteins.
  • a basic protein showing a positive charge is ionically bonded to a cation exchanger (carrier) having a negative charge, and an acidic protein showing a negative charge is bound to an anion exchanger having a positive charge, so that the protein is included.
  • the protein binds to the ion exchanger by passing the sample through a column packed with ion exchangers. After that, by increasing the salt concentration of the solvent passing through the column, the ionic bond between the protein and the ion exchanger becomes weaker, and the protein with the weakest binding force is sequentially removed from the ion exchanger and flows out of the column.
  • the selection of the cation exchanger or the anion exchanger shall be selected from the charge of the protein used as the sample.
  • Gel filtration chromatography This is a method for separating proteins by utilizing the difference in molecular weight of proteins.
  • the protein having a small molecular weight flows out while entering the small pores, and the protein having a large molecular weight flows out without entering the small pores. Therefore, the time for passing through the column is slow for proteins with a small molecular weight, and fast for proteins with a large molecular weight, so it is possible to separate proteins with a time lag.
  • Hydroxyapatite Chromatography Chromatography using hydroxyapatite, which is a kind of calcium phosphate. It is a method of separating proteins using multiple interactions, mainly based on metal affinity by calcium ions and cation exchange by phosphate groups. The carboxyl group and amino group of the amino acid are adsorbed by interacting with the carrier, respectively, and the target substance and the impurity are separated by flowing a solvent having a high concentration of phosphoric acid or a high salt concentration.
  • ⁇ Shaking Disintegration Test> The polymer-coated crosslinked alginate gel fiber or the multilayer polymer-coated crosslinked alginate gel fiber obtained by the above production method was suspended in phosphate-buffered physiological saline (PBS) and shaken for a certain period of time. Later, by confirming the susceptibility of the fiber to disintegrate (shaking disintegration degree), its physical strength can be measured. Specific test methods include, for example, the methods described in Examples described later.
  • ⁇ Tensile strength test> Using the polymer-coated crosslinked alginate gel fiber or the multilayer polymer-coated crosslinked alginate gel fiber obtained by the above manufacturing method, the breaking value (mN) is confirmed by using a tensile strength measuring device. , Its physical strength can be measured. Specific test methods include, for example, the methods described in Examples described later.
  • the strength of the polymer-coated cross-linked alginate gel fiber of the present invention is the static strength of the cross-linked alginate gel contained in the core layer constituting the fiber, and the cross-linked alginate gel formed between the core layer and the cationic polymer layer and the cationic polymer. This is due to the fact that the electrical action has optimum properties for the strength of the fibers of the present invention.
  • the strength of the multilayer polymer-coated crosslinked alginate gel fiber of the present invention is the crosslinked alginate gel contained in the core layer constituting the fiber, the crosslinked alginate gel formed between the core layer and the cationic polymer layer, and the static of the cationic polymer.
  • the electrical action and the electrostatic action of the cationic polymer and the anionic cationic polymer formed between the cationic polymer layer and the anionic polymer layer have the optimum properties for the strength of the fiber of the present invention. It is caused by the fact that it is.
  • the polymer-coated crosslinked alginate gel fiber or the multilayer polymer-coated crosslinked alginate gel fiber of the present invention has high physical stability, and the antibody, physiologically active substance, etc. produced in the core layer are released from the core layer, and further, the polymer layer is released. It also has appropriate permeability because it can permeate, and it is also a structure suitable for producing antibodies, physiologically active substances, and the like.
  • the reactive group or complementary reactive group introduction rate was introduced per uronic acid monosaccharide unit, which is a repeating unit of arginic acid. It means a value expressed as a percentage of the number of reactive groups or complementary reactive groups. In the examples below, the reactive group or complementary reactive group introduction rate (mol%) was calculated by the integral ratio of 1 H-NMR. The amount of alginic acid required to calculate the introduction rate is measured by the carbazole sulfate method using a calibration curve, and the amount of the reactive group or complementary reactive group is measured by the absorbance measurement method using a calibration curve. You can also do it.
  • the molecular weight of alginic acid into which a reactive group or a complementary reactive group has been introduced is bluedextran (molecular weight 2 million Da, SIGMA), tyroglobulin (molecular weight 66,900 Da, GE Healthcare Science), ferritin (molecular weight 2 million Da, SIGMA).
  • the amount of eluate of each component was plotted on the horizontal axis, and the logarithmic value of the molecular weight was plotted on the vertical axis, and linear regression was performed to create a calibration curve.
  • Two types of calibration curves were prepared, from blue dextran to ferritin and from ferritin to aprotinin.
  • the molecular weight (Mi) at the elution time i of the previously obtained chromatogram was calculated. Then, the absorbance at the elution time i was read and used as Hi. From these data, the weight average molecular weight (Mw) was calculated from the following formula.
  • the pattern of the NMR signal is s for singlet, d for doublet, t for triplet, q for quartet, m for multiplet, br for broad, J for coupling constant, Hz for hertz, CDCl 3 .
  • Means deuterated chloroform, DMSO - d 6 means deuterated dimethyl sulfoxide, and D2O means heavy water.
  • signals that cannot be confirmed because they are broadband, such as hydroxyl group (OH), amino group (NH 2 ), and carboxyl group (COOH) protons, are not described in the data.
  • M means molecular weight
  • RT means retention time
  • [M + H] + and [M + Na] + mean molecular ion peaks.
  • “Room temperature” or “rt” in the examples usually indicates a temperature of about 0 ° C to about 35 ° C.
  • “DMT-MM” in the examples is 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride (CAS REGISTRY NO .: 3945-69-). It means 5), and a commercially available product or a product synthesized by a method known in the literature can be used.
  • the rate of introduction of reactive groups (mol%) in the examples is calculated from the integral ratio of 1 H - NMR (D2O) and is introduced with respect to the number of moles of monosaccharides (gluronic acid and mannuronic acid) constituting alginic acid. It shall indicate the ratio of the number of moles of the reactive group obtained.
  • sodium alginate (A-1 to A-3 or B-2 to B-3) showing the physical property values shown in Table 8 was used as the sodium alginate.
  • sodium alginate or various alginate derivatives were sterilized by filtration as needed.
  • Tables 24-1 to 24-2 show the physical property values (specifically, the introduction of the reactive group) of the alginic acid derivative into which the reactive group was introduced, which were obtained in (Example 1) to (Example 18). The rate (mol%), molecular weight, and weight average molecular weight (Da)) are shown.
  • Tables 25-1 to 25-3 show 1 H-NMR and LC-Mass data of each intermediate in the examples.
  • the compounds 1-A2, 1-A1, 1-A3, 1-B2, 1-B2b, 1-B2c, 1-A2b, 1-A2c and 1-A2d were synthesized under the following synthesis method and reaction conditions. .. [Synthesis method] 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium in an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.) prepared in 1% by weight or 2% by weight. Chloride (DMT-MM), 1 molar concentration-sodium alginate was added.
  • DMT-MM dichloride
  • tert-butyl (4- (2- (2,2,2-trifluoroacetamide) ethoxy) benzyl) carbamate (IM-5-1): Commercially available tert-butyl (4-hydroxybenzyl) carbamate (formula RG5-1, CAS REGISTRY NO.: 14955-94-2) (0.36 g), N obtained by synthesizing commercially available or by a method known in the literature.
  • -(2-Bromoethyl) -2,2,2-trifluoroacetamide (formula SM5, CAS REGISTRY NO .: 75915-38-7) (0.46 g), potassium iodide (0.35 g) and N.
  • ⁇ Step4> Synthesis of N- (4- (2-aminoethoxy) benzyl-2- (cyclooct-2-in-1-yloxy) acetamide (IM5-4): (Example 5) Potassium carbonate (64.17 mg) and water (495 ⁇ L) were added to the mixture of the compound (99 mg) of the formula IM5-3 and methanol (1485 ⁇ L) obtained in ⁇ Step 3> under water-cooled stirring. ) was added, and the mixture was stirred at room temperature for 15 hours. After completion of the reaction, methanol was concentrated under reduced pressure, and the resulting aqueous layer was extracted 3 times with ethyl acetate (5 mL).
  • the compounds of 6-A2, 6-B2 and 6-B2b were synthesized under the following synthesis method and reaction conditions.
  • Synthesis method N- (2-aminoethyl) -2- (2-cyclooctyne) obtained by DMT-MM, a method known in the literature, in an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.) prepared to 1% by weight under room temperature stirring.
  • -1-Iroxy) -Acetamide [CAS REGISTRY NO. : 1809789-76-1] (SM6) in ethanol (EtOH 1) and 1 molar aqueous sodium hydrogen carbonate were sequentially added and stirred.
  • ⁇ Step1> Synthesis of tert-butyl (2-oxo-2-((2- (2,2,2-trifluoroacetamide) ethyl) amino) ethyl) carbamate (IM8-1): N- (2-Aminoethyl) -2,2,2-trifluoroacetamide hydrochloride (formula SM8) obtained by a method known in the literature [CAS REGISTRY NO. : 496946-73-7] (100 mg) and N- (tert-butoxycarbonyl) glycine (formula RG8-1) [CAS REGISTRY NO. : 4530-20-5] (91 mg) was dissolved in acetonitrile (3.0 mL).
  • ⁇ Step3> Synthesis of N- (2- (2- (2- (cyclooct-2-in-1-yloxy) acetamide) ethyl) -2,2,2-trifluoroacetamide (IM8-3): 2- (2-Cyclooctyne-1-iroxy) -acetic acid (formula RG5-2) obtained by a method known in the literature [CAS REGISTRY NO. : 917756-42-4] (80 mg), (Example 8) The compound (110 mg) of the formula IM8-2 obtained in ⁇ Step 2>, ethanol (1.6 mL), DMT-MM (219 mg).
  • Triethylamine (67 ⁇ L) was added, and the mixture was stirred at room temperature for 3 hours.
  • Water (3.2 mL) was added to the reaction mixture, the mixture was stirred at room temperature for 30 minutes, the solid was filtered, and the mixture was washed with water.
  • ⁇ Step4> Synthesis of N- (2- (aminoethyl) -2- (2- (cyclooct-2-in-1-yloxy) acetamide) acetamide (IM8-4): (Example 8) A solution of potassium carbonate (59 mg) in water (0.3 mL) was added to a solution of the compound (60 mg) of the formula IM8-3 obtained in ⁇ Step 3> in methanol (1.8 mL). , Stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure, water (2 mL) was added, and the mixture was saturated with sodium chloride.
  • ⁇ Step1> Synthesis of tert-butyl (3-oxo-3-((2- (2,2,2-trifluoroacetamide) ethyl) amino) propyl) carbamate (IM9-1): N- (2-aminoethyl) -2,2,2-trifluoroacetamide hydrochloride (formula SM8) (110 mg) and N- (tert-butoxycarbonyl) - ⁇ -alanine obtained by methods known in the literature. (Equation RG9-1) [CAS REGISTRY NO.
  • ⁇ Step2> Synthesis of 3-amino-N- (2- (2,2,2-trifluoroacetamide) ethyl) propanamide hydrochloride (IM9-2): (Example 9) After adding 4N hydrogen chloride / 1,4-dioxane (1.1 mL) under ice-water cooling to the compound (80 mg) of the formula IM9-1 obtained in ⁇ Step 1>, room temperature. Was stirred for 2 hours. Diisopropyl ether (3.4 mL) was added to the reaction mixture, and the mixture was stirred for 1.5 hours. The obtained solid was filtered to give the title compound (61 mg) as a white solid.
  • ⁇ Step3> Synthesis of 3- (2- (cyclooct-2-in-1-iroxy) acetamide) -N- (2- (2,2,2-trifluoroacetamide) ethyl) propanamide (IM9-3) : Ethanol (1.2 mL) was added to the compound of formula RG5-2 (44 mg) obtained by a method known in the literature and the compound of formula IM9-2 (61 mg) obtained by ⁇ Step 2> (Example 9). , DMT-MM (115 mg) and triethylamine (39 ⁇ L) were added, and the mixture was stirred at room temperature for 2 hours.
  • ⁇ Step4> Synthesis of N- (2- (aminoethyl) -3- (2- (cyclooct-2-in-1-yloxy) acetamide) propanamide (IM9-4): (Example 9) A solution of potassium carbonate (42 mg) in water (0.3 mL) is added to a solution of the compound (60 mg) of the formula IM9-3 obtained in ⁇ Step 3> in methanol (3.0 mL). After stirring at room temperature for 3 hours, a solution of potassium carbonate (42 mg) in water (0.3 mL) was further added, and the mixture was further stirred at room temperature for 16.5 hours.
  • ⁇ Step1> Synthesis of tert-butyl (2- (2- (3- (2,2,2-trifluoroacetamide) propanamide) ethoxy) ethyl) carbamate (IM10-1): A compound of formula SM10 obtained by a method known in the literature [CAS REGISTRY NO. : 50632-82-1] (400 mg), and a compound of formula RG10-1 obtained commercially available or by a method known in the literature (tert-butyl (2- (2-aminoethoxy) ethyl) carbamate, CAS REGISTRY NO.
  • ⁇ Step2> Synthesis of N- (2- (2-aminoethoxy) ethyl) -3- (2,2,2-trifluoroacetamide) propanamide hydrochloride (IM10-2): (Example 10) To the compound (451 mg) of the formula IM10-1 obtained in ⁇ Step 1>, 4N hydrogen chloride / 1,4-dioxane (3.16 mL) under ice-water cooling was added, and 3 at room temperature. Stir for hours. Diisopropyl ether (6.4 mL) was added to the reaction mixture, and the mixture was concentrated under reduced pressure to give the title compound (433 mg) as a colorless gum.
  • ⁇ Step4> Synthesis of 3-amino-N- (2- (2- (2- (cyclooct-2-in-1-yloxy) acetamide) ethoxy) ethyl) propanamide (IM10-4): (Example 10) To a solution of the compound (35 mg) of the formula IM10-3 obtained in ⁇ Step 3> in methanol (700 ⁇ L), a solution of potassium carbonate (33 mg) in water (175 ⁇ L) was added, and 16 at room temperature. . Stirred for 5 hours. The reaction mixture was concentrated under reduced pressure, water (2 mL) was added, and the mixture was saturated with sodium chloride.
  • ⁇ Step5> 3-Amino-N- (2- (2- (2- (cyclooct-2-in-1-yloxy) acetamide) ethoxy) ethyl) propanamide group-introduced alginic acid (10-A2) synthesis: DMT-MM (78 mg) in an aqueous solution (28 mL) of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd .: A-2) prepared to 1% by weight, (Example 10) Formula IM10-4 obtained by ⁇ Step 4>. A solution of the compound (24 mg) in ethanol (2.8 mL) and 1 molar concentration-sodium alginate (71 ⁇ L) was added.
  • Example 11a to l 4- (2-aminoethoxy) -N- (3-azidopropyl) benzamide group-introduced alginic acid (11-A2, 11-A1, 11-A3, 11-B2, 11-B2b, 11) -Synthesis of B2c, 11-A2b, 11-A2c, 11-B2d, 11-A2d, 11-A2e and 11-A3):
  • the compounds of 13-A2 and 13-A2b were synthesized by the following synthesis method and reaction conditions.
  • Synthesis method DMT-MM, a compound of formula SM13 synthesized by a method known in the literature (N- (2- (2-aminoethoxy) ethyl) -4, in an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.) prepared to 1% by weight.
  • -(Azidomethyl) benzamide hydrochloride; CAS REGISTRY NO .: 2401876-38-6) 1 molar concentration-hydrosodium aqueous solution was added and stirred.
  • the compounds of 14-A2, 14-B2 and 14-A2b were synthesized under the following synthesis method and reaction conditions.
  • DMT-MM a compound of formula SM14 synthesized by a method known in the literature (N- (2-aminoethyl) -4- (azidomethyl) benzamide) in an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.) prepared to 1% by weight.
  • Hydrochloride; CAS REGISTRY NO .: 2401876-25-1 1 molar concentration-sodium alginate was added and stirred. After adding sodium chloride, ethanol (EtOH 2) was added, and the mixture was stirred at room temperature.
  • Example 13-A2b the solid obtained by the above operation was dissolved in water and freeze-dried to obtain the title compound.
  • DMT-MM (112 mg) in an aqueous solution (40 mL) of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd .: A-2) prepared to 1% by weight, a compound of the formula SM15 synthesized by a method known in the literature (N- ( 2- (2- (2-Aminoethoxy) ethoxy) ethyl) -4- (azidomethyl) benzamide hydrochloride; CAS REGISTRY NO .: 2401876-41-1) (38 mg) in ethanol (4.0 mL), 1 molar concentration-bosodium solution (151 ⁇ L) was added, and the mixture was stirred at 30 ° C. for 3 hours.
  • the compounds of 16-A2 and 16-A2b were synthesized by the following synthesis method and reaction conditions.
  • Synthesis method DMT-MM, a compound of formula SM16 synthesized by a method known in the literature (N- (2- (2-aminoethoxy) ethyl) -4, in an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.) prepared to 1% by weight.
  • -Azidobenzamide hydrochloride CAS REGISTRY NO .: 2401876-47-7
  • 1 molar concentration-sodium alginate was added and stirred.
  • EtOH 2 ethanol
  • the obtained precipitate was collected by filtration, washed with ethanol, and dried under reduced pressure to give the title compound as a solid.
  • the solid obtained by the above operation was dissolved in water and freeze-dried to obtain the title compound.
  • the compounds of 17-B2, 17-B2b and 17-A2 were synthesized under the following synthesis method and reaction conditions.
  • DMT-MM a compound of formula SM17 synthesized by a method known in the literature (N- (2-aminoethyl) -4-azidobenzamide hydrochloride) in an aqueous solution of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd.) prepared to 1% by weight.
  • CAS REGISTRY NO .: 164013-00-7 1 molar concentration-sodium alginate was added and stirred. After adding sodium chloride, ethanol (EtOH 2) was added, and the mixture was stirred at room temperature.
  • Example 17c the solid obtained by the above operation was dissolved in water and freeze-dried to obtain the title compound.
  • DMT-MM (112 mg) in an aqueous solution (40 mL) of sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd .: A-2) prepared to 1% by weight, a compound of formula SM18 synthesized by a method known in the literature (N- 2- (2- (2-Aminoethoxy) ethoxy) ethyl) -4-azidobenzamide hydrochloride; CAS REGISTRY NO .: 2401876-48-8) (45 mg) in ethanol (4.0 mL), 1 mol. Concentration-bosodium solution (151 ⁇ L) was added and stirred at 30 ° C. for 3 hours.
  • Example F1-A Preparation of crosslinked alginate gel fiber (1) 3 wt% aqueous alkin solution (alkyn solution) prepared from compound 4-A2d or 1-A2d and 3 wt% aqueous azide solution (azid solution) prepared from compound 11-A2d, compound 13-A2b or compound 16-A2b. ) was used to mix the Alkin solution and the azide solution in equal volumes in the combinations shown in Table 26 to prepare a chemically modified arginic acid mixed solution (F1A-M1).
  • Example F1-B Preparation of crosslinked alginate gel fiber (2) An equal volume of a 3 wt% aqueous solution of 3 wt% compound 4-A2d prepared from compound 4-A2d and an aqueous solution of 3 wt% compound 11-A2d prepared from compound 11-A2d were mixed to obtain a mixed solution of chemically modified alginic acid (F1B-M1). Was prepared. A 3 wt% sodium alginate aqueous solution prepared from the mixed solution F1B-M1 and sodium alginate (B-2) was mixed at a volume ratio of 1/2 to obtain a 3 wt% alginate mixed solution (F1B-M1B).
  • -BS The concentration of blue dextran and the concentration of sodium chloride are as shown in Table 27) were mixed at the volume ratio shown in Table 27 to obtain an alginic acid mixed solution (F1B-M3).
  • the blue dextran concentration (mg / mL) and sodium chloride concentration (mg / mL) in the mixed solution F1B-M3 were adjusted so that blue dextran was 10 mg / mL and sodium chloride was 9 mg / mL.
  • the mixed solution F1B-M3 was filled in a Hamilton syringe. Subsequently, a metal needle (Musashi Engineering, SNA-19GB), a silicon tube (AS ONE, ⁇ 1 ⁇ ⁇ 2) and a glass capillary (Narikige, G-1) were sequentially connected to the syringe and set in the syringe pump. The tip of the glass capillary was immersed in a beaker containing a 100 mmol / L calcium chloride aqueous solution and injected at a flow rate of 250 ⁇ L / min for 1 minute. The fibrous substance injected into the calcium chloride aqueous solution was obtained as a crosslinked alginate gel fiber (CLA-1B) by allowing it to stand for 30 minutes or more (see CLA-1B No. in Table 27).
  • CLA-1B crosslinked alginate gel fiber
  • Example F1-C Preparation of polymer-coated crosslinked alginate gel fiber (1)
  • the crosslinked alginate gel fiber in the calcium chloride aqueous solution obtained in the above (Example F1-A) or (Example F1-B) was filtered and separated using a cell strainer.
  • the separated cross-linked alginate gel fiber was added to an aqueous solution containing a cationic polymer having each composition shown in Table 28, and the mixture was shaken and stirred at 37 ° C. and 125 rpm for 20 minutes to polymer-coat the cross-linked alginate gel fiber.
  • the fibers in the aqueous solution are filtered and separated using a cell strainer, washed twice with 5 mL of physiological saline, and polymer-coated crosslinked alginate gel fiber (CFB-1) (CFB-1 No. in Table 29). .) was obtained.
  • Example F1-D Stability of Polymer-Coated Crosslinked Alginate Gel Fiber (1) EDTA Treatment of Fiber
  • the polymer-coated crosslinked alginate gel fiber (CFB-1) obtained in the above (Example F1-C) was prepared with 20 mM EDTA. It was added to 2Na / physiological saline (5 mL), and the mixture was shaken and stirred at 37 ° C. and 125 rpm for 20 minutes.
  • the chelated polymer-coated crosslinked alginate gel fiber was again filtered using a cell strainer, separated, and washed twice with 5 mL of physiological saline.
  • the obtained polymer-coated crosslinked alginate gel fiber was immersed in 5 mL of physiological saline until the stability evaluation test was performed.
  • Stability assessment 3: No fiber disintegration / dissolution / deformation / elution of blue dextran, etc. 2: Disintegration / dissolution / deformation / elution of blue dextran (cumulative less than 100 ⁇ g / mL), etc. are observed in a part of the fiber 1 : Clear disintegration / dissolution / deformation / elution of blue dextran (cumulative 100 ⁇ g / mL or more), etc. are observed in the fiber.
  • Example F2-A Preparation of crosslinked alginate gel fiber containing antibody-producing cells
  • a G016 medium having the composition shown in Table 31 below was prepared.
  • methotrexate hereinafter referred to as MTX
  • MTX methotrexate
  • AGS2A 3 wt% sodium alginate aqueous solution (containing 0.9% sodium chloride) (ALGS2) or sodium alginate (A-2) prepared from the mixed solution F2A-M1 and sodium alginate (B-2) and 0.9 wt% sodium chloride aqueous solution.
  • AGS2A 3 wt% sodium chloride aqueous solution (containing 0.9% sodium chloride) (ALGS2A) prepared from a 0.9 wt% sodium chloride aqueous solution were mixed at the ratios shown in Table 34, and the alginic acid mixed solution (F2A-M2) was mixed. And said.
  • an antibody-producing medium solution containing the mixed solution F2A-M2 and anti-GPVI antibody-producing cells (2 ⁇ 107 cells / mL) was mixed in equal volumes to prepare a mixed solution (F2A-M3).
  • the mixed solution F2A-M3 was filled in a Hamilton syringe, a luer lock needle (Kanto Chemical Co., Inc., 15/23 NL-F) was connected to the syringe, and the mixture was set in a syringe pump.
  • the tip of the needle was immersed in a beaker containing a 100 mmol / L calcium chloride aqueous solution, and the needle was injected into the calcium chloride aqueous solution at a flow rate of 250 ⁇ L / min for 2 minutes.
  • the fibrous substance injected into the calcium chloride aqueous solution was allowed to stand for 30 minutes or more to obtain a crosslinked alginate gel fiber (CLA-G) containing anti-GPVI antibody-producing cells (CLA- in Table 34). See G No.).
  • Example F2-B Preparation of crosslinked alginate gel fiber containing physiologically active substance-producing cells A complete medium having the composition shown in Table 35 below was prepared.
  • the alkyne aqueous solution of F2A1 and the azide aqueous solution of F2N2 shown in Tables 32 and 33 of (Example F2-A) were mixed in equal volumes to prepare a chemically modified alginic acid mixed solution (F2B-M1).
  • F2B-M1 a chemically modified alginic acid mixed solution
  • a 3 wt% alginic acid aqueous solution (containing 0.9% sodium chloride) prepared from the mixed solution F2B-M1 and sodium alginate (B-2) and a 0.9 wt% sodium chloride aqueous solution was mixed at a ratio of 1: 2.
  • Alginic acid mixed solution (F2B-M2) Alginic acid mixed solution
  • the complete medium of Table 35 containing the mixed solution F2B-M2 and MIN6 cells (1 ⁇ 107 cells / mL) was mixed in equal volumes to obtain a mixed solution F2B-M3.
  • the mixed solution F2B-M3 was filled in a Hamilton syringe, a luer lock needle (Kanto Chemical Co., Inc., 15/23 NL-F) was connected to the syringe, and the mixture was set in a syringe pump.
  • the tip of the needle was immersed in a beaker containing a 100 mmol / L calcium chloride aqueous solution, and the needle was injected into the calcium chloride aqueous solution at a flow rate of 125 ⁇ L / min for 2 minutes.
  • the fibrous substance injected into the calcium chloride aqueous solution was obtained as a crosslinked alginate gel fiber (CLA-M) (FB2-B-1) containing MIN6 cells by allowing it to stand for 30 minutes or more.
  • CLA-M crosslinked alginate gel fiber
  • Example F2-C Preparation of Cell-Containing Polymer-Coated Crosslinked Alginate Gel Fiber
  • Example F2-A The various cell-containing crosslinked alginate gel fibers obtained in the above (Example F2-A) or (Example F2-B) are shown in Table 36.
  • Polymer coating cross-linking is performed by coating with a solution containing a cationic polymer of each composition in the same manner as described above (Example F1-C) (shaking stirring time is 30 minutes).
  • Alginate gel fiber (CFB-S) (see CFB-S No. in Table 37) was obtained.
  • Example F3 Confirmation of coating of crosslinked alginate gel fiber with a cationic polymer (Example F1-A) No.
  • a crosslinked alginate gel fiber (FB1-A-6) prepared under the conditions shown in F1-A-6, the mixture was immersed in an aqueous solution containing 0.1% poly-L-lysine-FITC label / 100 mM calcium chloride. .. After performing the same operation as in (Example F1-C), the surface of the obtained fiber was observed using a fluorescence microscope.
  • Example F4-A Preparation of crosslinked alginate gel fiber (3)
  • a 3 wt% alginic acid aqueous solution (containing 0.9 wt% sodium chloride) (ALGS2) was prepared from sodium alginate (B-2) and physiological saline for injection (manufactured by Otsuka Pharmaceutical Factory) (INs). Subsequently, an alkyne aqueous solution and an azide aqueous solution were prepared according to the formulations shown in Tables 38 and 39 below.
  • the alkyne aqueous solution (F4A1, F4A2) and the azide aqueous solution (F4N1, F4N2 and F4N3) were mixed in equal volumes according to the combinations shown in the table below to obtain a chemically modified alginic acid mixed solution (F4A-M1).
  • the mixed solution F4A-M1 and ALGS2 were mixed at a ratio of 1: 2 to obtain an alginic acid mixed solution (F4A-M2).
  • the mixed solution F4A-M3 is filled in a Hamilton syringe, and the syringe is filled with a metal needle (Musashi Engineering, SNA-19GB), a silicon tube (AS ONE, ⁇ 1 ⁇ ⁇ 2) and a glass capillary (Narikige, G-1). They were connected in sequence and set in a syringe pump.
  • the tip of the glass capillary was immersed in a beaker containing a 100 mmol / L calcium chloride aqueous solution, and the glass capillary was injected into the calcium chloride aqueous solution at a flow rate of 250 ⁇ L / min for 1 minute.
  • the fibrous substance injected into the calcium chloride aqueous solution was allowed to stand for 30 minutes or more to obtain a crosslinked alginate gel fiber (CLA-X1) (see CLA-X1 No. in Table 40).
  • Example F4-B Preparation of crosslinked alginate gel fiber (4)
  • the alkyne aqueous solution (F4A1) and the azide aqueous solution (F4N1) described in (Example F4-A) were mixed in equal volumes to obtain a chemically modified alginic acid mixed solution (F4B-M1).
  • the mixed solution F4B-M1 and the ALGS2 were prepared according to the formulations shown in Table 41 below, and used as an alginic acid mixed solution (F4B-M2).
  • a mixed solution (F4B-M3) was prepared at the mixing ratio of the alginic acid mixed solution F4B-M2 and INs shown in Table 41 below.
  • the mixed solution F4B-M3 is filled in a Hamilton syringe, and the syringe is filled with a metal needle (Musashi Engineering, SNA-19GB), a silicon tube (AS ONE, ⁇ 1 ⁇ ⁇ 2) and a glass capillary (Narikige, G-1). They were connected in sequence and set in a syringe pump.
  • the tip of the glass capillary was immersed in a beaker containing a 100 mmol / L calcium chloride aqueous solution, and the glass capillary was injected into the calcium chloride aqueous solution at a flow rate of 250 ⁇ L / min for 1 minute.
  • the fibrous substance injected into the calcium chloride aqueous solution was allowed to stand for 30 minutes or more to obtain a crosslinked alginate gel fiber (CLA-X2) (see CLA-X2 No. in Table 41).
  • Example F4-C Preparation of polymer-coated crosslinked alginate gel fiber (2)
  • the crosslinked alginate gel fibers (CLA-X1 and CLA-X2) obtained in the above (Example F4-A) or (Example F4-B) in an aqueous calcium chloride solution were filtered and separated using a cell strainer. ..
  • the separated cross-linked alginate gel fiber is added to an aqueous solution of 0.1% poly-L-ornithine hydrobromide / 100 mM calcium chloride, shaken and stirred at 37 ° C. and 125 rpm for 30 minutes, and the cross-linked alginate gel fiber is stirred.
  • Example F4-C Preparation of polymer-coated crosslinked alginate gel fiber (2)
  • the fibers in the aqueous solution are filtered and fractionated using a cell strainer, washed twice with 5 mL of saline, and polymer coated crosslinked alginate gel fiber (CFB-X) (CFB-X No. in Table 42). .) was obtained.
  • Example F4-D Tensile test of polymer-coated cross-linked alginate gel fiber
  • the tensile test of the polymer-coated cross-linked alginate gel fiber (CFB-X) obtained in the above (Example F4-C) is a small tabletop gel strength measuring machine.
  • EZ-SX 5NC1 Shiadzu, No. I308256D0592
  • the fiber was set on a jig and measured in physiological saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.). The measured values are shown in Table 43 in terms of the stress at the break of the fiber in MPa and the strain in%.
  • the culture was carried out for 14 or 20 days while shaking at rpm. Once every 2 to 3 days, 1.8 mL of the culture solution is withdrawn, 1.8 mL of the antibody-producing medium solution or 1.8 mL of the feed solution (Catalog No. JX F003, manufactured by Irvine) is added, and the total amount of the medium is 30. Keeped in mL. In addition, half of the culture solution was exchanged once a week. During the culture period, the IgG concentration of the culture medium was measured as a human IgG concentration by a Cedex Bio analyzer (Roche Diagnostics). In the culture using the cell-encapsulated polymer-coated crosslinked alginate gel fiber, the cumulative antibody amount during the culture period and the concentration of the anti-GPVI antibody-producing CHO cells detected in the culture medium were shown in Table 44.
  • a 60 mm ultra-low adhesive surface dish (Corning) is obtained by culturing a polymer-coated cross-linked alginate gel fiber containing MIN6 cells (Example F2-C). Incorporated, product number: 3261), the complete medium (5 mL) described in (Example F2-B) was added, and the mixture was allowed to stand in an incubator at 37 ° C. in a 5% CO 2 atmosphere for 3 days. Alternatively, it was cultured for 14 days.
  • MIN6 cell-encapsulated polymer-coated crosslinked alginate gel fiber was cultured in 10 mL of low glucose solution (2 mM glucose / KRBH / 0.1% BSA) for 2 hours and then in high glucose solution (20 mM glucose / KRBH / 0.1% BSA). After changing the solution to 10 mL, the cells were cultured for another 2 hours.
  • the solution was exchanged with 10 mL of low glucose solution again, and the cells were cultured for 2 hours.
  • the insulin concentration in solution at the end of each step was measured using an ultrasensitive mouse insulin measurement kit (manufactured by Morinaga Seigaku Kenkyusho). It was confirmed that insulin was released depending on the glucose concentration.
  • Example F5-A Preparation of crosslinked alginate gel fiber containing antibody-producing cells (Example F2-A) 3 of Compound 4-A2d and Compound 11-A2d in the same manner as in the formulations shown in Tables 32 and 33. After preparing a weight% aqueous solution (containing 0.9% by weight sodium chloride), each aqueous solution was mixed in equal volumes to prepare a chemically modified alginic acid mixed solution (F5A-M1).
  • alginic acid aqueous solution containing 0.9% sodium chloride
  • ALGS2 3 wt% alginic acid aqueous solution (containing 0.9% sodium chloride)
  • F5A-M1 3 wt% alginic acid aqueous solution (containing 0.9% sodium chloride)
  • F5A-M2 3 wt% alginic acid aqueous solution (containing 0.9% sodium chloride)
  • an equal volume of G016 medium having the composition described in (Example F2-A) containing the mixed solution F5A-M2 and tocilizumab-producing CHO cells (1 ⁇ 108 cells / mL) was mixed in equal volumes, and the cell-containing alginic acid mixed solution was mixed. It was designated as (F5A-M3).
  • the mixed solution F5A-M3 was filled in a Hamilton syringe, a luer lock needle (Kanto Chemical Co., Inc., 15/23 NL-F) was connected to the syringe, and the mixture was set in a syringe pump.
  • the tip of the needle is immersed in a beaker containing a 100 mmol / L calcium chloride aqueous solution or a 20 mmol / L barium chloride aqueous solution containing 0.9% sodium chloride, and in the calcium chloride or barium chloride aqueous solution at a flow rate of 250 ⁇ L / min.
  • a flow rate of 250 ⁇ L / min. was injected for 0.8 minutes.
  • the fibrous substance injected into the aqueous solution was allowed to stand for 30 minutes or more to obtain a crosslinked alginate gel fiber (CLA-G5) containing tocilizumab-producing CHO cells (CLA-G5 No. in Table 46). reference).
  • Example F5-B The composition of each of the antibody-producing cell-containing crosslinked alginate gel fibers (CLA-G5) obtained in the preparation of the antibody-producing cell-containing polymer-coated crosslinked alginate gel fiber (Example F5-A) is shown in Table 47.
  • Example F5-A The composition of each of the antibody-producing cell-containing crosslinked alginate gel fibers (CLA-G5) obtained in the preparation of the antibody-producing cell-containing polymer-coated crosslinked alginate gel fiber (Example F5-A) is shown in Table 47.
  • Table 47 was added to the cationic polymer-containing aqueous solution of the above, and the mixture was shaken and stirred at 37 ° C. and 125 rpm for 30 minutes to polymer-coat the cell-containing crosslinked alginate gel fiber.
  • the aqueous solution used for the polymer coating was 10 times the amount of the fiber to be coated.
  • Fibers in aqueous solution are filtered using a cell strainer, fractionated, washed twice with 5 mL of saline, and antibody-producing cell-containing polymer-coated cross-linked alginate gel fiber (CFB-S2) (in Table 48).
  • CFB-S2 No. was obtained.
  • Example FI-3 Culture of antibody-producing cell-containing polymer-coated cross-linked alginate gel fiber
  • FB5-B the antibody-producing cell-containing polymer-coated cross-linked alginate gel fiber obtained in (Example F5-B) (FB5).
  • the culture was started in the incubator while shaking at 125 rpm, and after 5 days, the culture temperature was set to 30 ° C., and the culture was continued at the same temperature. During this period, 1.8 mL of the culture medium is withdrawn once every 2 to 3 days, and 1.8 mL of the G016 medium or 1.8 mL of the feed solution (Catalog No. JX F003 manufactured by Irvine) is added to make the total amount of the medium 30. Keeped in mL. In addition, half of the culture solution was exchanged once a week. During the culture period, the IgG concentration of the culture medium was measured as a human IgG concentration by a Cedex Bio analyzer (Roche Diagnostics).
  • Table 49 shows the cumulative antibody concentration and the concentration of tosirizumab-producing CHO cells detected in the culture medium on each measurement day in the culture using the antibody-producing cell-encapsulated polymer-coated crosslinked alginate gel fiber. From the culture results, it was confirmed that the amount of antibody produced increased over time in the culture of each fiber.
  • Example F6 Preparation of antibody-producing cell-containing polymer-coated crosslinked alginate gel fiber
  • the description of Example F2-A which comprises tosirizumab-producing CHO cells (3 ⁇ 10 7 cells / mL or 1 ⁇ 10 8 cells / mL).
  • the G016 medium having the composition and the mixed solution F5A-M2 prepared in the above (Example F5-A) were mixed in equal volumes to obtain a cell-containing alginic acid mixed solution (F6A-M3).
  • the mixed solution F6A-M3 contained tocilizumab-producing CHO cells at the final concentrations of 0.5% by weight of the alkyne compound and the azide compound at the concentrations shown in Table 50.
  • the mixed solution F6A-M3 was filled in a Hamilton syringe, a luer lock needle (Kanto Chemical Co., Inc., 15/23 NL-F) was connected to the syringe, and the mixture was set in a syringe pump.
  • the tip of the needle was immersed in a beaker containing a 20 mmol / L barium chloride aqueous solution containing 0.9% sodium chloride, and injected into the aqueous solution at a flow rate of 250 ⁇ L / min for the time shown in Table 50.
  • the fibrous substance injected into the solution was allowed to stand for 30 minutes or more to obtain a crosslinked alginate gel fiber containing tocilizumab-producing CHO cells.
  • Example F5-B Subsequently described in (Example F5-B) above, using an aqueous solution containing 0.1% poly-L-alginate hydrobromide, 0.9% sodium chloride and 20 mM mmol / L barium chloride.
  • an antibody-producing cell-containing polymer-coated crosslinked alginate gel fiber (CFB-S3) (see CFB-S3 No. in Table 50) was obtained.
  • Example FI-4 Culture of antibody-producing cell-containing polymer-coated cross-linked alginate gel fiber
  • the antibody-producing cell-containing polymer-coated cross-linked alginate gel fiber obtained in (Example F6) (FB6-1) -C1, FB6--2-c1 or FB6-4-c1 is 1 bottle, FB6--3-c1 is 2 bottles), G016 medium having the composition described in (Example F2-A) is added, and a gel is added.
  • the total volume of the fiber and G016 medium was 30 mL, and the culture was started while shaking at 125 pm in the incubator under an atmosphere of 37 ° C.
  • the culture temperature was set to 30 ° C. and the culture was performed at the same temperature.
  • the culture temperature was set to 30 ° C. and the culture was performed at the same temperature.
  • 1.8 mL of the culture solution was withdrawn, 1.8 mL of the feed solution (Catalog No. JX F003 manufactured by Irvine) was added, and the total volume of the medium was kept at 30 mL. After that, half of the culture solution was exchanged once every 2 to 3 days.
  • the IgG concentration of the culture medium was measured as a human IgG concentration by a Cedex Bio analyzer (Roche Diagnostics).
  • Table 51 shows the cumulative antibody concentration and the concentration of tosirizumab-producing CHO cells detected in the culture medium on each measurement day in the culture using the antibody-producing cell-encapsulated polymer-coated crosslinked alginate gel fiber. From the culture results, it was confirmed that the amount of antibody produced increased over time in the culture of each fiber.
  • Example F7 Preparation of polymer-coated crosslinked alginate gel fiber containing antibody-producing cells
  • the mixed solution F5A-M2 prepared in Example F5-A) was mixed in equal volumes to obtain a cell-containing alginic acid mixed solution (F7A-M3).
  • the mixed solution F7A-M3 contained an alkyne compound and an azide compound at a final concentration of 0.5% by weight.
  • the mixed solution F7A-M3 was filled in a Hamilton syringe, a luer lock needle (Kanto Chemical Co., Inc., 15/23 NL-F) was connected to the syringe, and the mixture was set in a syringe pump.
  • the tip of the needle was immersed in a beaker containing a 20 mmol / L barium chloride aqueous solution containing 0.9% sodium chloride, and injected into the aqueous solution at a flow rate of 250 ⁇ L / min for the time shown in Table 52.
  • the fibrous substance injected into the aqueous solution was allowed to stand for 30 minutes or more to obtain a crosslinked alginate gel fiber containing anti-GPVI antibody-producing cells.
  • Example F5-B Subsequently described in (Example F5-B) above, using an aqueous solution containing 0.1% poly-L-alginate hydrobromide, 0.9% sodium chloride and 20 mM mmol / L barium chloride.
  • an antibody-producing cell-containing polymer-coated crosslinked alginate gel fiber (CFB-S4) (see CFB-S4 No. in Table 52) was obtained.
  • Example FI-5 Culture of antibody-producing cell-containing polymer-coated cross-linked alginate gel fiber
  • FB7-1 obtained in (Example F7) was placed.
  • -C1 or FB7-2-c1 is 1 bottle
  • FB7-3-c1 is 2 bottles
  • the antibody-producing medium solution described in (Example F2-A) is added to the gel fiber and antibody-producing medium solution.
  • the total volume was 30 mL, and the cells were cultured in an incubator at 37 ° C. under a 5% CO 2 atmosphere while shaking at 125 rpm.
  • Example F8 Preparation of polymer-coated cross-linked alginate gel fiber containing antibody-producing cells 3 wt% alginic acid aqueous solution (0.) From various sodium alginate and saline for injection (manufactured by Otsuka Pharmaceutical Factory) (INs) according to the formulation shown in Table 54 below. Contains 9 wt% sodium chloride). Subsequently, a 3 wt% alkyne aqueous solution and an azide aqueous solution (containing 0.9 wt% sodium chloride) were prepared according to the formulations shown in Table 55 below.
  • the 3 wt% alkyne aqueous solution and the azide aqueous solution (containing 0.9 wt% sodium chloride) shown in Tables 54 and 55 were mixed in equal volumes according to the formulation shown in Table 56 below, and the chemically modified arginic acid mixed solution (F8A-M1) was mixed.
  • the mixed solution F8A-M1 and the above-mentioned 3 wt% sodium alginate aqueous solution (containing 0.9 wt% sodium chloride) are mixed in the combination shown in Table 56 below so as to be 5:10, and the alginic acid mixed solution (F8A-M2) is mixed. ) Was prepared.
  • the G016 medium having the composition described in (Example F2-A) containing tosirizumab-producing CHO cells (1 ⁇ 10 8 cells / mL) and the mixed solution F8A-M2 were mixed in equal volumes, and the cell-containing alginic acid mixed solution was mixed. It was designated as (F8A-M3).
  • the mixed solution F8A-M3 contained an alkyne compound and an azide compound at a final concentration of 0.5% by weight.
  • the mixed solution F8A-M3 was filled in a Hamilton syringe, a luer lock needle (Kanto Chemical Co., Inc., 15/23 NL-F) was connected to the syringe, and the mixture was set in a syringe pump.
  • the tip of the needle was immersed in a beaker containing a 20 mmol / L barium chloride aqueous solution containing 0.9% sodium chloride, and injected at a flow rate of 250 ⁇ L / min for 0.8 minutes.
  • the fibrous substance injected into the aqueous solution was allowed to stand for 30 minutes or more to obtain a crosslinked alginate gel fiber containing tocilizumab-producing CHO cells.
  • Example FI-6 Culture of antibody-producing cell-containing polymer-coated cross-linked alginate gel fiber
  • the antibody-producing cell-containing polymer-coated cross-linked alginate gel fiber obtained in (Example F8) (FB8-1) -C1, FB8-2-c1, FB8-3-c1, FB8-4-c1 or FB8-5-c1) is added and a G016 medium (30 mL) having the composition described in (Example F2-A) is added.
  • the IgG concentration of the culture medium was measured as a human IgG concentration by a Cedex Bio analyzer (Roche Diagnostics).
  • Table 57 shows the cumulative antibody concentration and the concentration of tosirizumab-producing CHO cells detected in the culture medium on each measurement day in the culture using the antibody-producing cell-encapsulated polymer-coated crosslinked alginate gel fiber. From the culture results, it was confirmed that the amount of antibody produced increased over time in the culture of each fiber.
  • Example F9 Preparation of antibody-producing cell-containing polymer-coated crosslinked alginate gel fiber
  • the G016 medium having the composition according to (Example F2-A) containing tosirizumab-producing CHO cells (1 ⁇ 108 cells / mL) and the above (Example F9).
  • the alginic acid mixed solution F5A-M2 prepared in Example F5-A) was mixed in equal volumes to obtain a cell-containing alginic acid mixed solution (F9A-M3).
  • the final concentration of the alkyne compound and the azide compound was contained in the mixed solution F9A-M3 in an amount of 0.5% by weight.
  • the mixed solution F9A-M3 was filled in a Hamilton syringe, a luer lock needle (Kanto Chemical Co., Inc., 15/23 NL-F) was connected to the syringe, and the mixture was set in a syringe pump.
  • the tip of the needle was immersed in a beaker containing a 20 mmol / L barium chloride aqueous solution containing 0.9% sodium chloride, and injected into the aqueous solution at a flow rate of 250 ⁇ L / min for 0.8 minutes.
  • the fibrous substance injected into the solution was allowed to stand for 30 minutes or more to obtain a crosslinked alginate gel fiber containing tocilizumab-producing CHO cells.
  • the obtained cell-containing crosslinked alginate gel fiber is coated with the cationic polymer-containing aqueous solution having each composition shown in Table 58 in the same manner as described in the above (Example F5-B).
  • a polymer-coated crosslinked alginate gel fiber (CFB-S6) containing antibody-producing cells was obtained.
  • Example FI-7 Culture of antibody-producing cell-containing polymer-coated cross-linked alginate gel fiber
  • FB9- the antibody-producing cell-containing polymer-coated cross-linked alginate gel fiber obtained in the above (Example F9) (FB9-).
  • the culture was started while shaking at 125 pm in the incubator, and after 5 days, the culture temperature was set to 30 ° C., and the culture was continued at the same temperature. During this period, 1.8 mL of the culture solution is withdrawn once every 2 to 3 days, 1.8 mL of the G016 medium or 1.8 mL of the feed solution (Catalog No. JX F003 manufactured by Irvine) is added, or half of the culture solution is replaced. Was carried out. During the culture period, the IgG concentration of the culture medium was measured as a human IgG concentration by a Cedex Bio analyzer (Roche Diagnostics).
  • Table 60 shows the cumulative antibody concentration and the concentration of tosirizumab-producing CHO cells detected in the culture medium on each measurement day in the culture using the antibody-producing cell-encapsulated polymer-coated crosslinked alginate gel fiber. From the culture results, it was confirmed that the amount of antibody produced increased over time in the culture of the fibers of FB9-1-c1 and FB9-1-c3.
  • Example F10 Preparation of cross-linked alginate gel fiber coated with multilayer polymer ⁇ Step a> Preparation of cross-linked alginic acid gel fiber 1 and 20 mg / mL blue dextran (Cytiva, Blue Dextran 2000) prepared to 3.0% by weight of alginic acid aqueous solution, Blue Dextran 2000, Code No. 17036001) Equal amounts of 1.8 wt% saline containing were mixed and filled into a Hamilton syringe.
  • a metal needle (Musashi Engineering Metal Needle, SNA-19GB), a silicon tube (AS ONE, ⁇ 1 ⁇ ⁇ 2) and a glass capillary (Narikige, G-1) were sequentially connected to the syringe and set in the syringe pump.
  • the tip of the glass capillary was immersed in a 100 mmol / L calcium chloride aqueous solution, and the solution was sent at a flow rate of 250 ⁇ L / min for 1 minute.
  • the recovered crosslinked alginate gel fiber was allowed to stand in an aqueous solution of calcium chloride (10 mL) having the same concentration for 30 minutes.
  • the alginic acid aqueous solution 1 used in step a is an aqueous solution of sodium alginate (A-2) or a mixed solution of compound 4-A2c / compound 11-A2e.
  • ⁇ Step b> PLO coating (Example F10)
  • the crosslinked alginate gel fiber was separated from the calcium chloride aqueous solution containing the crosslinked alginate gel fiber obtained in ⁇ step a> using a cell strainer. Separated cross-linked alginate gel fiber prepared to 0.1% by weight of poly-L-ornithine (PLO) aqueous solution (5 mL) [0.1% poly-L-ornithine hydrochloride and 100 mmol / L calcium chloride is contained.
  • PLO poly-L-ornithine
  • Aqueous solution was added, and the mixture was shaken and stirred at 37 ° C. and 125 rpm for 20 minutes. Then, the cationic polymer-coated crosslinked alginate gel fiber was separated from the poly-L-ornithine aqueous solution using a cell strainer, and washed twice with 5 mL of physiological saline.
  • ⁇ Step c> Alginic acid coating (Example F10)
  • the PLO-coated cationic polymer-coated crosslinked alginic acid gel fiber obtained in ⁇ Step b> was added to alginic acid aqueous solution 2 (5 mL) prepared to 0.15% by weight, and 37 The mixture was shaken and stirred at 125 rpm for 20 minutes at ° C. Then, using a cell strainer, the multilayer polymer-coated crosslinked alginic acid gel fiber (CFB-10) (see CFB-10 No. in Table 61 below) was separated from the alginic acid aqueous solution, and twice using 5 mL of physiological saline. Washed.
  • the alginate aqueous solution 2 used in step c is an aqueous solution of sodium alginate (A-2) or a mixed solution of compound 4-A2c / compound 11-A2e.
  • Example F10B Preparation of Multilayer Polymer Coated Crosslinked Alginate Gel Fiber Using a 3% by weight aqueous solution of compound 4-A2d, a 3% by weight aqueous solution of compound 11-A2d, and an aqueous solution of sodium alginate (A-2), as shown in Table 62 below.
  • An alginic acid solution (F10B-M1) was prepared with the combination shown. Subsequently, a 1.8% aqueous sodium chloride solution containing 20 mg / mL blue dextran (Blue Dextran 2000, GE Healthcare Science, 17036001) and F10B-M1 were mixed in equal volumes to prepare an alginic acid mixed solution (F10B-M2).
  • the mixed solution F10B-M2 was contained so that the final concentration of the alkyne compound and the azide compound was 1.5% by weight or the final concentration of sodium alginate was 1.5% by weight.
  • the tip of the glass capillary was immersed in a beaker containing a 20 mmol / L barium chloride aqueous solution containing 0.9% sodium chloride, and injected into the solution at a flow rate of 250 ⁇ L / min for 1 minute.
  • the fibrous substance injected into the solution was allowed to stand in a solution of the same concentration for 30 minutes to obtain a crosslinked alginate gel fiber.
  • the solution containing the obtained crosslinked alginate gel fiber was filtered using a cell strainer, and the gel fiber was separated.
  • the separated gel fiber is added to a cationic polymer-containing aqueous solution (5 mL) containing 0.1% poly-L-alginic acid hydrochloride, 0.9% sodium chloride and 20 mM mmol / L barium chloride, and added to 37 ° C. , Shaking and stirring at 125 rpm for 20 minutes to obtain a cationic polymer coated crosslinked alginate gel fiber.
  • the obtained gel fiber was filtered again using a cell strainer and washed twice with 5 mL of physiological saline. Subsequently, sodium alginate (A-2) was dissolved in physiological saline to prepare a final concentration of 0.15% alginate solution (F10B-M3).
  • the washed cationic polymer-coated crosslinked alginate gel fiber was added to F10B-M3 (5 mL), and the mixture was shaken and stirred at 37 ° C. and 125 rpm for 20 minutes to obtain a multilayer polymer-coated crosslinked alginate gel fiber (CFB-10B) (Table 62 below).
  • CFB-10B multilayer polymer-coated crosslinked alginate gel fiber
  • Medium CFB-10B No. was obtained.
  • the obtained CFB-10B was filtered using a cell strainer and washed twice with 5 mL of physiological saline.
  • Example F11 Stability of Multilayer Polymer Coated Crosslinked Alginate Gel Fiber ⁇ Step d> Chelate Treatment (Example F10) Multilayer Polymer Coated Crosslinked Alginate Gel Fiber obtained in ⁇ Step c> (CFB-10 No. in Table 63 below) (See.) was added to 20 mM EDTA. 2K / physiological saline (5 mL), and the mixture was shaken and stirred at 37 ° C. and 125 rpm for 20 minutes. Then, the multilayer polymer-coated crosslinked alginate gel fiber was separated from EDTA.2K / saline using a cell strainer, and washed twice with 5 mL of saline.
  • the recovered multilayer polymer-coated crosslinked alginate gel fiber (see CFB-11 No. in Table 63 below) was immersed in 5 mL physiological saline until the stability evaluation test was performed.
  • ⁇ Step e> Stability evaluation (Example F11)
  • the multilayer polymer-coated crosslinked alginate gel fiber obtained in ⁇ Step d> was transferred to a 15 mL centrifuge tube containing 5 mL of PBS solution and shaken at 37 ° C. for 1 day (water bath). Shaker (manufactured by TAITEC, personal II), 180 rpm). The fiber was then transferred to a 25 mL centrifuge tube containing 10 mL of PBS solution and shaken at 37 ° C. for an additional hour.
  • the observation results are shown in Table 63 below.
  • Stability assessment (score): 3: No fiber disintegration / dissolution / deformation / elution of blue dextran, etc. 2: Disintegration / dissolution / deformation / elution of blue dextran, etc. are observed in a part of the fiber 1: Clear disintegration / dissolution in the fiber / Deformation / Elution of blue dextran, etc. is observed, and the function of the structure is not maintained.
  • Example F11B Stability of Multilayer Polymer Coated Crosslinked Alginate Gel Fiber (1) EDTA Treatment of Fiber Multilayer Polymer Coated Crosslinked Alginate Gel Fiber (CFB-10B) produced in the above (Example F10B) (CFB- in Table 64 below). By carrying out the same operation as in (Example F11) using 10B No.), a chelated multilayer polymer-coated crosslinked alginate gel fiber (CFB-11B) (CFB-11B No. in Table 64 below). See).
  • Example F12 Preparation of cross-linked alginic acid gel fiber coated with a multilayer polymer containing antibody-producing cells
  • Alginic acid mixed solution F5A-M2 prepared in the above example (Example F5-A) and saline solution for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) (INs) were mixed at a ratio of 1:19 to prepare an anionic polymer-containing aqueous solution (the anionic polymer-containing aqueous solution contained 0.15% by weight of F5A-M2).
  • the antibody-producing cell-containing cationic polymer-coated cross-linked alginate gel fiber (FB9-1-c1, FB9-2-c2 or FB9-3-c3) obtained by the method described in Example F9 was used as a fiber 10 It was immersed in a double amount of the anionic polymer-containing aqueous solution, stirred at 37 ° C. and 125 rpm for 20 minutes, and coated with the anionic polymer. Fibers in aqueous solution are filtered using a cell strainer, separated, washed 3 times with 7 mL of INs, and cross-linked alginate gel fiber (ACFB-S1) containing antibody-producing cells and coated with a multilayer polymer (ACFB- in Table 65). (See S1 No.) was obtained.
  • ACFB-S1 cross-linked alginate gel fiber
  • Example FI-8 Culturing of cross-linked alginate gel fiber coated with multi-layer polymer coated with antibody-producing cells
  • FB12 multi-layer polymer coated with antibody-producing cells
  • Example F13-A Preparation of cross-linked alginate gel fiber coated with cation polymer coated with antibody-producing cells Using tosirizumab-producing CHO cells, the same operations as described above (Example F5-A) and (Example F5-B) were carried out. By doing so, an antibody-producing cell-containing cationic polymer-coated crosslinked alginate gel fiber (CLA-G13) (see Table 67 CLA-G13 No. below) was prepared.
  • CLA-G13 contained tocilizumab-producing CHO cells at a final concentration of 5 ⁇ 107 cells / mL.
  • Example F13-B Preparation of multi-layer polymer-coated crosslinked alginate gel fiber containing antibody-producing cells
  • a 3 wt% alkyne aqueous solution and an azide aqueous solution (containing 0.9 wt% sodium chloride) were prepared according to the formulations shown in Tables 68 and 69 below. ..
  • sodium alginate (B-2) and physiological saline for injection were mixed, and a 3 wt% sodium alginate aqueous solution (containing 0.9% sodium chloride) (ALGS2) was added to sodium alginate (ALGS2).
  • A-2) and INs were mixed to prepare a 3 wt% sodium alginate aqueous solution (containing 0.9% sodium chloride) (ALGS2A).
  • the alkyne, azide, ALGS2, ALGS2A or INs were mixed in the combination shown in Table 70 to prepare an anionic polymer-containing aqueous solution.
  • the total concentration of alginic acid contained in the anionic polymer-containing aqueous solution was about 0.15% by weight, and the mixture was mixed so as to have the ratios shown in Table 70.
  • CLA-G13 cationic polymer-coated crosslinked alginate gel fiber
  • Example FI-9 Culturing of antibody-producing cell-containing multilayer polymer-coated cross-linked alginate gel fiber
  • Example F13-B Culturing of antibody-producing cell-containing multilayer polymer-coated cross-linked alginate gel fiber
  • FB13-B-1-ac1, FB13-B-2-ac2, FB13-B-3-ac2, FB13-B-4-ac2, FB13-B-5-ac2, FB13-B-6-ac2, FB13 -B-7-ac2 and FB13-B-8-ac2) were added, the G016 medium (30 mL) described in (Example F2-A) was added, and the temperature was 37 ° C. under a 5% CO 2 atmosphere.
  • the culture was started in the incubator while shaking at 125 rpm, and after 5 days, the culture temperature was set to 30 ° C., and the culture was continued at the same temperature.
  • 1.8 mL of the culture medium is withdrawn once every 2 to 3 days, and 1.8 mL of the G016 medium or 1.8 mL of the feed solution (Catalog No. JX F003 manufactured by Irvine) is added to make the total amount of the medium 30. Keeped in mL. In addition, half of the culture solution was exchanged once a week.
  • the IgG concentration of the culture medium was measured as a human IgG concentration by a Cedex Bio analyzer (Roche Diagnostics).
  • Table 71 shows the cumulative antibody concentration and the concentration of tosirizumab-producing CHO cells detected in the culture medium on each measurement day in the culture using the antibody-producing cell-containing polymer-coated crosslinked alginate gel fiber.
  • Example F14 Confirmation of coating of crosslinked alginate gel fiber with anionic polymer / cationic polymer 3 weights of alkyne compound 4-A2d and azide compound 11-A2d in the same manner as in the formulation described in (Example F2-A).
  • A% aqueous solution (containing 0.9% by weight sodium chloride) was prepared.
  • a chemically modified arginic acid mixed solution (F14A-M1) was prepared by equally mixing 3% by weight alkyne and an aqueous azide solution (containing 0.9% by weight sodium chloride).
  • a 3 wt% alginic acid aqueous solution (containing 0.9% sodium chloride) (ALGS2) prepared from the mixed solution F14A-M1 and sodium alginate (B-2) and saline for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) (INs).
  • the aqueous solution F14A-M3 was filled in a Hamilton syringe, a luer lock needle (Kanto Chemical Co., Inc., 15/23 NL-F) was connected to the syringe, and the mixture was set in a syringe pump.
  • the tip of the needle was immersed in a beaker containing a 20 mmol / L barium chloride aqueous solution containing 0.9% sodium chloride, and injected at a flow rate of 250 ⁇ L / min for 0.8 minutes.
  • the fibrous substance injected into the solution was allowed to stand for 30 minutes or more to obtain a crosslinked alginate gel fiber.
  • Example F9 the same operation as in (Example F9) (the aqueous solution used for coating was 2 mL) was carried out to obtain a cationic polymer-coated crosslinked alginate gel fiber.
  • the composition of the cationic polymer-containing aqueous solution used for coating is as shown in Table 72 below, and 2 mL was used for each fiber.
  • the fluorescently labeled alginic acid (LA) synthesized from sodium alginate (A-2) and fluorescein-5-thiosemicarbazide (CAS REGISTORY No .: 76863-28-0) was adjusted to a final concentration of 0.15% by weight.
  • LAPS anionic polymer-containing aqueous solution
  • the cationic polymer coated crosslinked alginate gel fiber was immersed in 2 mL of LAPS, stirred at 37 ° C. and 125 rpm for 20 minutes, and coated with an anionic polymer.
  • the fibers in aqueous solution are filtered using a cell strainer, fractionated, washed once with 8 mL of saline and anion-cationic polymer crosslinked alginate gel fiber (LCLA-G) (LCLA in Table 73). -G No.) was obtained.
  • the surface of the obtained fiber was observed using a fluorescence microscope. The observation results are shown in FIGS. 22 and 23. It was confirmed that the surface of the gel fiber was coated with LA (the color inverted in the background of FIGS. 22 and 23 is LA).
  • Example F15-A Preparation of polymer-coated crosslinked alginate gel fiber 3 wt% alginic acid aqueous solution (0.9 wt% chloride) from sodium alginate (B-2) and saline for injection (manufactured by Otsuka Pharmaceutical Factory) (INs) Sodium-containing) (ALGS2) was prepared. Subsequently, an alkyne aqueous solution and an azide aqueous solution were prepared according to the formulations shown in Table 74 below. F15A1, F15N1 and ALGS2 were mixed in the combinations and ratios shown in Table 75 below to prepare a 3 wt% alginic acid solution (F15A-M1).
  • a mixed solution (F15A-M2) was prepared by mixing F15A-M1 and INs in equal volumes.
  • the total concentration of alginic acid contained in F15A-M2 is 1.5% by weight.
  • the tip of the glass capillary was immersed in a beaker containing a 100 mmol / L calcium chloride aqueous solution and injected at a flow rate of 250 ⁇ L / min for 0.8 minutes.
  • the fibrous substance injected into the aqueous solution was allowed to stand for 30 minutes or more to obtain a crosslinked alginate gel fiber.
  • the obtained crosslinked alginate gel fiber was filtered using a cell strainer, separated, and added to an aqueous solution containing 0.1% poly-L-ornithine hydrobromide and 100 m mmol / L calcium chloride at 37 ° C.
  • the crosslinked alginate gel fiber was coated with a cationic polymer by shaking and stirring at 125 rpm for 30 minutes.
  • the aqueous solution used for the polymer coating was 10 times the amount of the fiber to be coated.
  • the fibers in the aqueous solution are filtered and separated using a cell strainer, washed 3 times with 7 mL of physiological saline, and polymer-coated crosslinked alginate gel fiber (CFB-G15A) (CFB-G15A No. in Table 75). .) was obtained.
  • Example F15-B Preparation of Multilayer Polymer Coated Crosslinked Alginic Acid Gel Fiber
  • the F15A-M1 was diluted with INs to a total alginic acid concentration of 0.15% by weight with the composition shown in Table 76 below, and an anion was added.
  • An aqueous solution containing a sex polymer was prepared.
  • the polymer-coated crosslinked alginate gel fiber (FB15-A-1-c1 or FB15-A-2-c1) obtained in Example F15-A was added to the anionic polymer-containing aqueous solution, and the same as in (Example F12).
  • a multilayer polymer-coated crosslinked alginate gel fiber (ACFB-G15B) (see ACFB-G15B No. in Table 76) was obtained by carrying out the coating operation of.
  • Example F15-C Tensile test of multilayer polymer coated crosslinked alginate gel fiber
  • the tensile test of the multilayer polymer coated crosslinked alginate gel fiber (CFB-G15B) obtained in the above (Example F15-B) is a small tabletop gel strength.
  • EZ-SX 5NC1 Shiadzu, No. I308256D0592
  • the measured values are shown in Table 77 as the stress at the break of the fiber in MPa and the strain in%.
  • a polymer-coated crosslinked alginate gel fiber in which a core layer containing cells capable of producing an antibody, a physiologically active substance, etc. and a crosslinked alginate gel is coated with a cationic polymer (cationic polymer layer) is provided. Further, it is possible to provide a method for producing the fiber and a method for culturing an antibody, a physiologically active substance, or the like using the fiber. Further, the core layer containing cells capable of producing antibodies, physiologically active substances, etc. and a crosslinked alginate gel is coated with a cationic polymer (cationic polymer layer) and an anionic polymer (anionic polymer layer). Gel fibers are provided. Further, it is possible to provide a method for producing the fiber and a method for culturing an antibody, a physiologically active substance, or the like using the fiber.
  • a Polymer-coated cross-linked arginate gel fiber or multilayer polymer-coated cross-linked arginate gel fiber core layer diameter
  • b Polymer-coated cross-linked arginate gel fiber or multi-layer polymer-coated cross-linked arginate gel fiber cationic polymer layer thickness
  • c Polymer-coated cross-linked Outer diameter of alginate gel fiber
  • d Multilayer polymer coating outer diameter
  • e Multilayer polymer coating
  • Crosslinked alginate gel fiber anionic polymer layer thickness 4 Cationic polymer layer 5: Core layer 6: Cell (antibody) , Cells that can produce physiologically active substances, etc.) 7: Anionic polymer layer
  • XX Equipment YY: Extruder cylinder 1: Introduction port 2: Discharge port
  • DD Container (for example, beaker) (divalent metal ion-containing solution)
  • EE Container (eg, beaker) (cationic polymer-containing solution)
  • FF Container (eg, bea

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
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Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993013136A1 (en) 1991-12-20 1993-07-08 Howmedica Inc. Ultra-pure polysaccharide materials for medical use
JPH08269102A (ja) 1995-03-30 1996-10-15 Shiseido Co Ltd エンドトキシンフリーのβ１，３−グルカン及びその製造法並びに医療用ゲル素材
US5589591A (en) 1986-07-03 1996-12-31 Advanced Magnetics, Inc. Endotoxin-free polysaccharides
JPH09324001A (ja) 1996-04-02 1997-12-16 Kyowa Hakko Kogyo Co Ltd ヒアルロン酸ナトリウムの精製法
JP2005036036A (ja) 2003-07-16 2005-02-10 Tanabe Seiyaku Co Ltd エンドトキシン除去方法
JP2007075425A (ja) 2005-09-15 2007-03-29 Mochida Pharmaceut Co Ltd アルギン酸を含む創傷被覆材
WO2011046105A1 (ja) 2009-10-14 2011-04-21 国立大学法人 東京大学 被覆されたマイクロゲルファイバ
JP2014506926A (ja) * 2011-03-04 2014-03-20 ウェイク・フォレスト・ユニヴァーシティ・ヘルス・サイエンシズ ホルモン補充療法のためのカプセル化された細胞
JP2014136128A (ja) 2013-01-18 2014-07-28 Univ Of Tokyo 移植用神経束及びその製造方法
JP2014236698A (ja) 2013-06-07 2014-12-18 国立大学法人 鹿児島大学 細胞凝集塊作製法
WO2015178427A1 (ja) 2014-05-20 2015-11-26 国立大学法人 東京大学 中空マイクロファイバ
JP2016508369A (ja) * 2013-02-08 2016-03-22 シャンハイ インスティチューツ フォー バイオロジカル サイエンシーズ、 チャイニーズ アカデミー オブ サイエンシーズ ヒト肝細胞様細胞およびその使用
JP2016077229A (ja) 2014-10-17 2016-05-16 国立大学法人 東京大学 ファイバ状基材、３次元細胞構造体及びその製造方法、並びに３次元細胞構造体の培養方法
WO2019078251A1 (ja) 2017-10-19 2019-04-25 国立大学法人東京大学 接着剤及びその使用
JP2019075993A (ja) 2017-10-20 2019-05-23 国立大学法人 東京大学 細胞の三次元培養方法、細胞構造体、細胞構造体の製造方法及び細胞の運搬方法
WO2019123886A1 (ja) 2017-12-23 2019-06-27 国立大学法人東京大学 アレイ及びその使用
JP2019520837A (ja) * 2016-07-18 2019-07-25 イーティーエイチ・チューリッヒ Β細胞ミメティック細胞
WO2019240219A1 (ja) 2018-06-14 2019-12-19 持田製薬株式会社 新規な架橋アルギン酸
WO2020032221A1 (ja) 2018-08-10 2020-02-13 持田製薬株式会社 アルギン酸中空マイクロファイバ
WO2021125279A1 (ja) 2019-12-18 2021-06-24 持田製薬株式会社 化学架橋アルギン酸ゲルファイバ
WO2021125255A1 (ja) 2019-12-18 2021-06-24 持田製薬株式会社 新規な架橋アルギン酸

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH031931A (ja) 1989-05-30 1991-01-08 Toshiba Seiki Kk 加熱シール装置
US9090868B2 (en) * 2010-07-12 2015-07-28 President And Fellows Of Harvard College Alginate hydrogel fibers and related materials
US11655343B2 (en) * 2016-03-24 2023-05-23 Takeda Pharmaceutical Company Limited Alginate hydrogel compositions

Patent Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5589591A (en) 1986-07-03 1996-12-31 Advanced Magnetics, Inc. Endotoxin-free polysaccharides
WO1993013136A1 (en) 1991-12-20 1993-07-08 Howmedica Inc. Ultra-pure polysaccharide materials for medical use
JPH08269102A (ja) 1995-03-30 1996-10-15 Shiseido Co Ltd エンドトキシンフリーのβ１，３−グルカン及びその製造法並びに医療用ゲル素材
JPH09324001A (ja) 1996-04-02 1997-12-16 Kyowa Hakko Kogyo Co Ltd ヒアルロン酸ナトリウムの精製法
JP2005036036A (ja) 2003-07-16 2005-02-10 Tanabe Seiyaku Co Ltd エンドトキシン除去方法
JP2007075425A (ja) 2005-09-15 2007-03-29 Mochida Pharmaceut Co Ltd アルギン酸を含む創傷被覆材
WO2011046105A1 (ja) 2009-10-14 2011-04-21 国立大学法人 東京大学 被覆されたマイクロゲルファイバ
JP2014506926A (ja) * 2011-03-04 2014-03-20 ウェイク・フォレスト・ユニヴァーシティ・ヘルス・サイエンシズ ホルモン補充療法のためのカプセル化された細胞
JP2014136128A (ja) 2013-01-18 2014-07-28 Univ Of Tokyo 移植用神経束及びその製造方法
JP2016508369A (ja) * 2013-02-08 2016-03-22 シャンハイ インスティチューツ フォー バイオロジカル サイエンシーズ、 チャイニーズ アカデミー オブ サイエンシーズ ヒト肝細胞様細胞およびその使用
JP2014236698A (ja) 2013-06-07 2014-12-18 国立大学法人 鹿児島大学 細胞凝集塊作製法
WO2015178427A1 (ja) 2014-05-20 2015-11-26 国立大学法人 東京大学 中空マイクロファイバ
JP2016077229A (ja) 2014-10-17 2016-05-16 国立大学法人 東京大学 ファイバ状基材、３次元細胞構造体及びその製造方法、並びに３次元細胞構造体の培養方法
JP2019520837A (ja) * 2016-07-18 2019-07-25 イーティーエイチ・チューリッヒ Β細胞ミメティック細胞
WO2019078251A1 (ja) 2017-10-19 2019-04-25 国立大学法人東京大学 接着剤及びその使用
JP2019075993A (ja) 2017-10-20 2019-05-23 国立大学法人 東京大学 細胞の三次元培養方法、細胞構造体、細胞構造体の製造方法及び細胞の運搬方法
WO2019123886A1 (ja) 2017-12-23 2019-06-27 国立大学法人東京大学 アレイ及びその使用
WO2019240219A1 (ja) 2018-06-14 2019-12-19 持田製薬株式会社 新規な架橋アルギン酸
WO2020032221A1 (ja) 2018-08-10 2020-02-13 持田製薬株式会社 アルギン酸中空マイクロファイバ
WO2021125279A1 (ja) 2019-12-18 2021-06-24 持田製薬株式会社 化学架橋アルギン酸ゲルファイバ
WO2021125255A1 (ja) 2019-12-18 2021-06-24 持田製薬株式会社 新規な架橋アルギン酸

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
"ASTM F2064-00", 2006, ASTM INTERNATIONAL
"Comprehensive Organic Transformations", 2018, article "A Guide to Functional Group Preparations"
"Strategic Applications of Named Reactions in Organic Synthesis", 2005, ACADEMIC PRESS
APPL MICROBIOL BIOTECHNOL, vol. 40, 1994
CAS , no. 2401876-48-8
CAS, no. 127828-22-2
CHIKAKO YOMOTA, BULL. NATL. HEALTH SCI., vol. 117, 1999, pages 135 - 139
CHIKAKO YOMOTA, BULL. NATL. INST. HEALTH SCI., vol. 121, 2003
EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, no. 6, 2014, pages 1280 - 1286
GREENE: "The Fifth Series of Experimental Chemistry", 2007, MARUZEN CO., LTD.
HASAN ULUDAG , PAUL DE VOS , PATRICK A TRESCO: "Technology of mammalian cell encapsulation", ADVANCED DRUG DELIVERY REVIEWS, ELSEVIER, AMSTERDAM , NL, vol. 42, no. 1-2, 20 August 2000 (2000-08-20), Amsterdam , NL , pages 29 - 64, XP002302229, ISSN: 0169-409X, DOI: 10.1016/S0169-409X(00)00053-3 *
MASUMI YAMADA, SUGAYA SARI, NAGANUMA YOJI, SEKI MINORU: "Microfluidic synthesis of chemically and physically anisotropic hydrogel microfibers for guided cell growth and networking", SOFT MATTER, vol. 8, no. 11, 3 February 2012 (2012-02-03), GB , pages 3122 - 3130, XP055553869, ISSN: 1744-683X, DOI: 10.1039/c2sm07263g *
ORG. PROCESS RES. DEV., vol. 22, 2018, pages 108 - 110
ORGANOMETALLICS, vol. 29, no. 23, 2010, pages 6619 - 6622
PA-LAB CHIP, vol. 8, 2008, pages 1255 - 1257
See also references of EP4268857A4
STAHLWERMUTH: "Handbook of Pharmaceutical Salts: Properties, Selection, and Use", 2002, WILEY-VCH

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4361249A4 (en) * 2021-06-23 2025-09-24 Mochida Pharm Co Ltd NEW POLYMER-COATED CROSSLINKED ALGINATE GEL FIBER

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